JPH107671A - Anti-thrombogenic diamide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a specific anti-thrombogenic diamide selectively exhibiting an inhibiting action to thrombin in a short time independent of an antithrombin III and useful for an anti coagulation agent, etc., without interrupting dissolution of blood clot keeping hemostasis. SOLUTION: This new anti-thrombogenic diamide is expressed by the formula I [X-C(O) is D-prolinyl, D-homoprolinyl, the formula: R<m> -(CH2 )g -NH-CH2 -C(O) R<m> is a carboxyl ester or an SO2 (1-4C) alkyl, etc.; (g) is 1, 2 or 3], the formula II T is a 3-8C cycloalkyl, a 1-8C alkyl, phenyl or naphthyl, etc.; R' is H, a 1-4C alkyl, a 1-4C fluoroalkyl or an aryl, etc.; (a) is 0, 1 or 2}, etc.; Y-C(O) is a group of the formula: NR<g> -CH2 C(O) (R<g> is a 1-6C alkyl, a 3-8C cycloalkyl or an aryl, etc.) or the formula III (r) is 0, 1 or 2}, etc.; Im is a (substituted) imidazol-4-yl, etc.], and selectively exhibits an inhibiting action to thrombin in a short time, and is useful for a curing agent of a thromboembolism disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a thrombin inhibitor useful as an anticoagulant in mammals.
More particularly, the present invention relates to diamides having high anticoagulant and antithrombotic activities. Accordingly, the present invention provides novel inhibitors of thrombin, pharmaceutical compositions containing the compounds as active ingredients, and thromboembolism such as venous thrombosis, pulmonary embolism, arterial thrombosis (particularly myocardial ischemia, heart failure and cerebral thrombosis) As anticoagulants of the compounds for the prevention and treatment of general and localized hypercoagulable conditions associated with inflammatory processes such as inflammatory diseases, angioplasty and coronary artery bypass grafts and total tissue trauma Regarding the use of In addition, the diamide is useful as an anticoagulant in in vitro applications.

[0002]

BACKGROUND OF THE INVENTION The process of blood coagulation, or thrombus formation, is triggered by a complex proteolytic cascade that leads to the formation of thrombin. Thrombin proteolytically removes the activating peptide from the Aα and Bβ chains of fibrinogen, which is soluble in plasma, and initiates insoluble fibrin formation. Currently, heparin and coumarin are administered to achieve anticoagulation. Parenteral pharmacological control of coagulation and thrombosis is based on the inhibition of thrombin by the use of heparin. Heparin acts indirectly on thrombin by promoting the inhibitory effect of endogenous antithrombin III (a major physiological inhibitor). Heparin cannot be an effective treatment because antithrombin III concentrations are altered in plasma and surface-bound thrombin appears to be resistant to this indirect mechanism. The heparin concentration must be monitored by a coagulation assay since the efficacy and safety are likely to be related to the coagulation assay (in particular, activated partial thromboplastin time (AP
TT) Assay). Coumarin is a post-translational gamma in the synthesis of prothrombin and other proteins of this type.
Blocking carboxylation prevents thrombin generation. Due to the mechanism of action, the effects of coumarin only appear as late as 6 to 24 hours after administration. Furthermore, they are not selective anticoagulants. Coumarin also requires monitoring by a coagulation assay (especially the prothrombin time (PT) assay). Recently, there has been interest in small synthetic molecules that exhibit strong direct inhibition on thrombin. For example, Robert M. Scarborough's Annual Report in Medicinal Chemistry (199
See 5), 30, 71-80. Although heparin and coumarin are effective anticoagulants, no commercial drug from small synthetic molecules has yet emerged; and, despite continued promising potential for this class of compounds, they selectively respond to thrombin. Of anticoagulant drugs that act, independently of antithrombin III, have an inhibitory effect shortly after administration (preferably oral administration) and do not interfere with the dissolution of clots required to maintain hemostasis. The need still exists.

[0003]

It is required to act selectively on thrombin, to exert an inhibitory action shortly after administration (preferably oral administration) independently of antithrombin III, and to maintain hemostasis. To provide an anticoagulant that does not interfere with the dissolution of the clot that is to be obtained.

[0004]

The present invention is based on the discovery that the compounds of the present invention as defined below are potent thrombin inhibitors with high bioavailability through oral administration. The present invention provides a compound of formula (I): X-C ( O) -Y-C (O) -NH-CH 2 -G-Im I [ wherein, X-C (O) - is, D- prolinyl, D - _{^{homoprolinyl, R m - (CH 2)}} g -NH-CH 2 -C (O)
−,

Embedded image [Where R ^d is carboxy or methylsulfonyl;
R ^e is NHR ^c , NHCOR ^c or NHCOOR ^c (where R ^c is (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₈ ) cycloalkyl or (C ₃ -C ₈ ) having 4 to 10 carbon atoms. Cycloalkyl- (C ₁ -C ₆ ) alkyl); T is (C ₃ -C ₈ ) cycloalkyl, (C ₁ -C ₈ ) alkyl,

Embedded image _a is 0, 1 or 2; and Q is -OH, (C ₁ -C ₄₎
Alkoxy or —NH—A; A is hydrogen, (C ₁ -C ₄ )
_{Alkyl, R "SO 2 -, R} " OC (O) -, R "C (O)
^{-, R n C (O)} - or ^{_{- (CH 2) g -R m}} ; g is 1,
2 or 3; B is hydrogen or (C ₁ -C ₄ ) alkyl;
R 'is hydrogen or (C ₁ -C ₄₎ alkyl; R "is (C ₁ -
C ₄ ) alkyl, having ₁ to 5 fluorine atoms (C _1-
C _{4) fluoroalkyl, - (CH 2) d -R} m , or unsubstituted or substituted aryl (wherein aryl is phenyl,
Naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocycle having 1 or 2 heteroatoms selected from the same or different sulfur, oxygen and nitrogen, or the same or different selected from sulfur, oxygen and nitrogen 9 or 10 members having one or two heteroatoms
Unsubstituted or substituted fused bicyclic aromatic heterocycle); R
^m is -COOR ^b , -SO ₂ (C ₁ -C ₄ ) alkyl, -S
_{O 3 H, -P (O)} (OR b) 2 or tetrazol -5
- yl; R ⁿ is -COOR ^b or tetrazol-5-yl; each R ^b independently hydrogen or (C ₁ -C ₄₎ alkyl; _d is 1, 2 or 3; ^m is 0, 1 or 2; And Z
Is hydrogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkoxy, hydroxy, halo or (C ₁ -C ₄ ) alkylsulfonylamino];

Embedded image[Where R^gIs (C₁-C₆) Alkyl, (C_Three-C₈)
Cycloalkyl or-(CH_Two)_p-L- (CH2)_q
-T '; R^pIs (C₁-C₆) Alkyl, (C_Three-C₈) Shiku
Loalkyl or-(CH_Two)_p-L- (CH2)_q-T '
(here,_pIs 0, 1, 2, 3, or 4; L is a single bond,
-O-, -S- or -NH-;_qIs 0, 1, 2 or
3; and T ′ is (C₁-C _Four) Alkyl, (C_Three-C₈)
Chloroalkyl, -COOH, -CONH_TwoOr Ar
(Where Ar is unsubstituted or substituted aryl (where
Aryl is phenyl, naphthyl, same or different
One or two heliums selected from sulfur, oxygen and nitrogen
5- or 6-membered unsubstituted or substituted terrorist atom
Aromatic heterocycle, or sulfur or acid, same or different
One or two heteroatoms selected from nitrogen and nitrogen
9- or 10-membered unsubstituted or substituted fused bicyclic ring
Formula aromatic heterocycle)); R^yIs -CH_Two-, -O-, -S-
Or -NH-; and R^zIs a single bond or R^yAnd 3
Two adjacent carbon atoms taken together to saturate 5-8 atoms
When forming a carbocycle, one of those atoms is -O
-, -S- or -NH-; and_rIs
0, 1 or 2] G is-(CH_Two)_s− (Where_sIs 0, 1, 2, 3 or
Is 4), or G is-(CH_Two)_t-CH = CH- (here
so,_tIs 0, 1 or 2 and the double bond is trans
And Im has R at position 5.
Where R is hydrogen, hydro
May be substituted with a xy substituent (C₁-C_FourA)
Alkyl or C 2-4 (C₁-C_Three) Alkoxy-
(C₁-C_Three) Alkyl)] or
Provides a pharmaceutically acceptable salt thereof.

[0005] The present invention further provides a pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient. The present invention also provides a method for suppressing thrombosis in a mammal, which comprises administering an antithrombin amount of the compound I to the mammal in need of treatment. The present invention further provides a method for inhibiting thrombin, which comprises administering a thrombin-inhibiting amount of Compound I to a mammal in need of treatment. The present invention relates to a novel inhibitor of thrombin, a pharmaceutical composition containing the compound as an active ingredient, and thromboembolic diseases such as venous thrombosis, pulmonary embolism, and arterial thrombosis (particularly myocardial ischemia, heart failure and cerebral thrombosis). ,
It relates to the use of said compounds as anticoagulants for the prevention and treatment of general and local hypercoagulable conditions associated with inflammatory processes such as angioplasty and coronary artery bypass grafting and total tissue trauma .

[0006] The definitions of the terms in this specification are as follows unless otherwise specified. "Halo"
It is fluoro, chloro, bromo or iodo. “Alkyl”, “alkoxy” and the like refer to both straight-chain and branched-chain groups; however, certain groups such as “propyl” refer only to the straight-chain group and branched groups such as “isopropyl”. Expressed separately as “5- or 6-membered aromatic heterocycle” means 1 or 2 nitrogen atoms; 1 sulfur atom;
Any one of a 5-membered or 6-membered ring having a stable structure containing one oxygen atom; one nitrogen atom and one sulfur atom; or one nitrogen atom and one oxygen atom. . A five-membered ring has two double bonds and a six-membered ring has three double bonds. “9- or 10-membered fused bicyclic aromatic heterocycle”
Is defined as any of the condensed rings formed by ortho-condensation of either the 5-membered or 6-membered ring to the benzene ring or the 6-membered heteroaromatic ring in such a manner as to take a stable structure. Also means. The above heterocycle and imidazole moiety I
Many of m can exist in tautomeric forms.
All such isomers are included within the scope of the present invention.

Each aromatic or heteroaromatic group described in the definition of Ar or R ″ above is independently unsubstituted or halo, hydroxy, (C ₁ -C ₄ ) alkyl, (C _1- C ₄ ) alkoxy, amino, mono (C ₁ -C ₄₎
Alkyl) amino, di (C ₁ -C ₄ alkyl) amino,-
(CH ₂ ) _j COOH, mercapto, —S (O) _h (C ₁ −
C _{4 alkyl), - NHS (O) h} (C 1 -C 4 alkyl), - NHC (O) ( C 1 -C 4 alkyl), - S
(O) _h NH ₂ , —S (O) _h NH (C ₁ -C ₄ alkyl)
Or —S (O) _h N (C ₁ -C ₄ alkyl) ₂ (where
_h is 0, 1 or 2; _j is 0, 1, 2, 3 or 4
Are independently substituted so as to have a stable structure with one or two substituents selected from

In the compounds of the formula I, X
The carbonyl function of the-(CO)-group is -Y- (CO)-
To the amine function of Then, -Y- (CO)-
Bond to the amino group shown in Formula I. As used herein, a group wherein both Z and A are hydrogen:

Embedded image Is sometimes referred to as phenylglycyl, and is abbreviated as Phg. For example, a compound in which A is methyl is Nα-methyl-phenylglycyl, abbreviated as MePhg. Z
Is, for example, 3′-chlorophenylglycyl or Phg (3Cl), depending on the type and position of the substituent. As used herein, a group wherein both Z and A are hydrogen:

Embedded image Is sometimes referred to as phenylalanyl, and is abbreviated as Phe. For example, a compound in which A is methyl is Nα-methyl-phenylalanyl, abbreviated as MePhe. Z
Is a compound other than hydrogen, for example, 3′-chlorophenylalanyl or Phe (3Cl), depending on the type and position of the substituent.

A group wherein R ′ is hydrogen:

Embedded image May also be referred to as 1- and 3-tetrahydro-isoquinolinecarbonyl, respectively, 1-Tig and 3-
Abbreviated as Tig. Groups wherein R ′ is hydrogen:

Embedded image May also be referred to as 1- and 3-perhydro-isoquinolinecarbonyl, respectively, 1-Pig and 3-P
ig. Although various condensed isomers of these substituents exist, as indicated by the wavy lines, the present invention contemplates any individual isomer and combinations thereof. a group wherein r is 0, 1 or 2:

Embedded image Is referred to as acetylidine-2-carbonyl, prolinyl or homoprolinyl, and is abbreviated as Azt, Pro or hPro.

Group:

Embedded image Represents a saturated bicyclic system of the 4,5; 5,5; 6,5; 7,5; or 8,5 type. The stereochemistry of 3a is cis to the carbonyl; other bridgehead bonds can be either cis or trans, except that the 4,5 and 5,5 systems must be cis at the bridgehead. The definitions of R ^y and R ^z provide that the variable ring is a saturated carbocyclic ring system of 4 to 8 carbon atoms, including the 3 carbon atoms shown. All ring atoms may be carbon, or one ring atom may be -O-, -S- and-
It may be a hetero atom selected from NH-. This definition includes:

Embedded image And a group derived from octahydroindole-2-carboxylic acid. The various cis and trans forms of this group are contemplated by the present invention.
The preferred isomer derived from [2S (2α, 3aβ, 7aβ)]-octahydroindole-2-carboxylic acid is abbreviated as Ohi and has the formula:

Embedded image Indicated by The asterisk in the Y group means the asymmetric center which is (L). The asterisk in group X is (D) or (DL)
Means a chiral center; # in the group X is (L)
Means an asymmetric center.

It will be appreciated that compound I exists in the form of tautomers or cis or trans isomers, optically active racemates or diastereomers and may be isolated. The present invention includes both tautomers and mixtures thereof. The invention encompasses compounds I as mixtures of diastereomers as well as individual diastereomers; the invention encompasses both mixtures of enantiomers as well as individual enantiomers; and mixtures or each of the forms It will be appreciated that both have inhibitory properties against thrombin, and methods for making and isolating specific forms and methods for determining the inhibitory properties against thrombin by the standard tests described below are known in the art. Further, compound I may exhibit polymorphism or may be a solvate with water or an organic solvent. The present invention also includes any of such polymorphs, solvates or mixtures thereof.

The following specific examples and ranges for groups and substituents are given for illustrative purposes only and exclude other values for the groups and substituents or other values within the defined ranges. Not something. (C ₁ -C ₄ ) alkyl,
Particular examples of (C ₁ -C ₆ ) alkyl, (C ₁ -C ₈ ) alkyl or (C ₁ -C ₁₀ ) alkyl are methyl,
Ethyl, propyl, isopropyl, butyl, isobutyl or t-butyl. Particular examples of (C ₁ -C ₄ ) alkoxy are methoxy, ethoxy, propoxy or isopropoxy. Specific examples of (C ₃ -C ₈₎ cycloalkyl is cyclopropyl, cyclopentyl or cyclohexyl. Particular examples of (C ₁ -C ₄ ) fluoroalkyl groups are trifluoromethyl or 2,2,2
-Trifluoroethyl. Particular examples of aryl are
Phenyl, naphthyl, furyl, thienyl, pyridyl, indolyl, quinolinyl or isoquinolinyl.
(C ₁ -C ₃₎ alkoxy - specific examples of (C ₁ -C ₃₎ alkyl is methoxymethyl, ethoxymethyl, methoxyethyl or ethoxyethyl.

[0013] Particular mention compounds ^{I, X a -C (O) -Y} a -C (O) -NH-CH 2 -G a -Im a Ia [ wherein, X ^a -C (O) - is, D-homoprolinyl,

Embedded image [Here, T ^a is cyclohexyl or phenyl; _a is 0, 1 or 2; and A ^a is hydrogen, (C ₁ -C ₄₎ alkyl, (C ₁ -C ₄₎ sulfonyl, (C ₁ -C ₄ alkyl )
Oxy - carbonyl, (C ₁ -C ₄ alkyl) carbonyl or ^{carbomethoxy]; - Y a -C (O} ) - is,

Embedded image [Here, _r is 0, 1 or 2]; G ^a is methylene, ethylene, trimethylene or trans kink two den; and Im ^a is imidazol-4-yl having a methyl or hydroxymethyl substituent at the 5-position] Or a pharmaceutically acceptable salt thereof.

A preferred compound Ia is

Embedded image Wherein T ^a is cyclohexyl or phenyl; _a is 1;
And A ^a is hydrogen, ethylsulfonyl or carboxymethyl; -Y ^a -C (O)-is

Embedded image [Wherein, _r is 0, 1 or 2]; G ^a is methylene, ethylene or trans kink two den; and Im ^a compound represented by a is] 4-imidazolyl or 5-methyl-imidazol-4-yl or a It is a pharmaceutically acceptable salt.

Further particularly preferred compounds Ia are

Embedded image Wherein T ^a is cyclohexyl; _a is 1; and A ^a is hydrogen, ethylsulfonyl or carboxymethyl;
^a -C (O)-is

Embedded image [Where _r is 1]; G ^a is ethylene or trans-vinylidene; and I m ^a is 4-imidazolyl] or a pharmaceutically acceptable salt thereof.

Particularly preferred compounds of the invention are those of Example 1,
The compounds described in 2, 7, 8, 10 or 15; more particularly preferred compounds are the compounds described in Example 7 or 8; or a pharmaceutically acceptable salt thereof. Compound I can be prepared by methods including the methods for producing structural analogs known in the chemical arts or by the novel methods described herein. The novel methods of preparation and intermediates of compound I further provide features of the present invention and are illustrated by the following procedures (generic group definitions are as described above unless otherwise specified). It will be appreciated that it is preferred or necessary to protect the functional group with a conventional protecting group to produce Compound I and then remove the protecting group to give Compound I.

(A) To prepare a compound I wherein G is-(CH ₂ ) _s- and _s is 2, 3 or 4, G is-
(CH ₂ ) _t —CH ＝ CH—, where _t is 0, 1 or 2
Is hydrogenated to the corresponding double bond of compound I.
For example, as described in Example 2, the acid addition salt of compound I dissolved in aqueous ethanol is hydrogenated at room temperature and pressure under a palladium / carbon catalyst. (B) Acid II: X—C (O) —Y—C (O) —OH II or its activated derivative is coupled with amine III: H ₂ N—CH ₂ —G-Im III. The coupling may be performed using a mixed anhydride method as described in Example 1 or a method using a reagent such as bis (2-oxo-oxazolidinyl) phosphine acid chloride (BOP-Cl) as described in Example 10. Alternatively, a conventional procedure such as a method using a reagent such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide as described in Example 16 is performed. (C) Acid IV: X—C (O) —OH IV or an activated derivative thereof is coupled with amine V: H—Y—C (O) —NH—CH ₂ —G—Im V. Coupling is performed using conventional procedures, such as using one of the methods described in (B) above. Thereafter, in any of the above procedures, if the functional group is protected using a protecting group, the protecting group is removed. Thereafter, in any of the above procedures, if a pharmaceutically acceptable salt of Compound I is required, the acid or base of Compound I is reacted with a base or acid that provides a physiologically acceptable counterion. Alternatively, it can be obtained by other conventional procedures such as exchange with a counter ion of a salt.

Compounds corresponding to compounds I in which one or more functional groups are protected provide another aspect of the invention. Such compounds have the formula ^{Ip: (P X) X-} C (O) - (P Y) can be expressed as Y-C (O) -NH- CH 2 -G-Im (P I) Ip, One or more protecting groups,
P ^X, is bound to P ^Y and P ^I. Where P ^X is the functional group: X-C (O) - any protection against group; P ^Y is the functional group: Y-C (O) - optional protecting groups for; and P ^I
Is an optional protecting group for the functional group: Im. Representative examples of P ^X and P ^Y include t-butyl ester or benzyl ester when the protected functional group is a carboxyl group, and t-butyl ester when the protected functional group is an amino group. A group that forms butyl urethane or benzyl urethane, and a group that forms methyl ether, t-butyl ether or benzyl ether when the protected functional group is a hydroxy group. Representative examples of P ^I is N- tosyl group protecting the N-H group of the imidazole. It will be appreciated that one compound I is a protected equivalent of another compound I. For example, as described in Example 1, compound I where A is R "OC (O)-[where R" is t-butyl] is a protected equivalent of compound I where A is hydrogen. It is. Similarly, R ^m is -COO
Compound I, which is R ^b [where R ^b is t-butyl]
^m is a protected equivalent of a compound I a and R ^b is hydrogen and -COOR ^b.

As noted above, the present invention includes pharmaceutically acceptable salts of the thrombin inhibiting compounds defined by Formula I. Certain diamide compounds of the present invention are useful for reacting with any of a number of inorganic and organic acids that provide non-toxic anions to form pharmaceutically acceptable salts.
It has one or more sufficiently basic functional groups. .
Acids commonly used to form acid addition salts include:
Inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, and p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid and citric acid And organic acids such as benzoic acid and acetic acid. Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate,
Metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, Propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, butyne-1,4-dioate, hexyne-1,4-
Diacid, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, Phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2 -Sulfonates, mandelates and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric, hydrobromic and sulfuric acids. In compounds I, wherein X or Y has an acid group such as a carboxy group, form a pharmaceutically acceptable salt with a base that provides a pharmaceutically acceptable cation, for example, an alkali metal salt (particularly sodium and Potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts and salts prepared from physiologically acceptable organic bases such as triethylamine, morpholine, piperidine and triethanolamine, and the like. .

The starting materials required for the preparation of the compounds I according to the invention are, if not commercially available, procedures selected from standard techniques in organic chemistry, such as aromatic and heteroaromatic nucleus substitution and transformation, known in the art. It can be produced by a procedure selected from the same techniques as those for the synthesis of structurally similar compounds, particularly peptide compounds, and a procedure selected from the above procedures or techniques similar to the procedures described in the Examples. It will be apparent to those skilled in the art that various methods can be applied to the preparation of the starting materials. The novel starting materials provide another aspect of the present invention.

The starting acid of formula II can also be represented by the formula IIp: (P ^x ) X—C (O) — (P ^Y ) Y—C (O) —OH IIp P ^X is any protecting group as defined above. The acid IIp can be prepared by converting an optionally protected acid of the formula VI: (P ^x ) XC (O) —OH VI to a compound of the formula VII: H— (P ^Y ) YC (O) —OP ^C VII [where P ^C is hydrogen or methyl, ethyl, t- butyl or a carboxy protecting group such as benzyl] in is an amino acid derivative and the coupling indicated, then if the protecting group P ^C is present, the protecting group It can be manufactured by removing. The amine V can be an N-protected amino acid of the formula VIII: (P ^N ) -YC (O) -OH VIII where ^PN is an amino protecting group, ) And then the protecting group P ^N
Can be prepared; for example, by the method described in Example 16.

The optionally protected amine III is
It can be prepared by a number of routes, including, for example, Scheme I wherein IIIp represents an optionally protected amine III. Reaction Scheme I

Embedded image Thus, alcohol X [where N of imidazole is N
-H converts the hydroxy group is conveniently protected] by N- tosyl group represented by P ^I in the leaving group, compound XI [wherein, Lg is, for example bromo, iodo, mesylate or tosylate And the like are leaving groups]. Then, using the compound XI, wherein metallated protected amine XII [, M is lithium, a metal ion such as sodium or potassium, N = N ^P etc. phthalimido group or a di -t- butyl iminodicarboxylate group Alkylation of the nitrogen of which represents an amino with a protecting and activating group gives the protected amine XIII. The protecting group P ^P and amine IIIp by [wherein removing Then, N-
Tosyl group P ^I get removed may be] Before coupling the amine be left. As an alternative, nitrile XIV may be reduced to give amine IIIp. [Wherein, G is - (CH _{2) t} -CH = a CH-, _t is 0, 1 or 2] double bond and cyano group of a nitrile XIV amine III [wherein corresponding simultaneously reducing And _s is 2, 3 or 4. Also, as described in Example 18, by substituting the leaving group with a cyanide ion, compound XI can be converted to the homologous compound X
It can also be converted to IV.

The compounds of the present invention are most efficiently isolated in the form of acid addition salts. Salts of Compound I formed with acids as described above are useful as pharmaceutically acceptable salts for the administration of antithrombin drugs and for the formulation of such drugs. Other acid addition salts may be prepared, isolated, and purified. As noted above, optically active isomers and diastereomers of Compound I of the present invention are also considered to be part of the present invention.
Such optically active isomers can be produced from the respective optically active precursors by the operation as described above, or by resolving a racemic mixture.
This resolution can be achieved by derivatization with a chiral reagent followed by chromatography or repeated crystallization. The chiral auxiliary is removed by standard methods to give a substantially optically pure isomer of the compound of the invention or a precursor thereof. A more detailed description of optical resolution can be found in Jax et al., Enantiomers, Ra.
cemates, and Resolutions, John Wiley and Sons (1981).

The compounds of the present invention selectively inhibit thrombin in a manner distinct from other protease and non-enzymatic compounds contained in coagulated blood, without interfering with the body's natural ability to dissolve blood clots. Low inhibitory effect on fibrinolysis). Further, due to such selectivity, the compounds of the present invention can also be used as thrombolytic agents that do not substantially interfere with thrombolysis and fibrinolysis. One aspect of the present invention is a method for inhibiting thrombin in a mammal, comprising administering to a mammal in need of treatment an effective amount of the compound I of the present invention in inhibiting thrombin. In another aspect of the present invention, the present invention provides a method for treating a mammal in need thereof, which comprises administering an effective amount of the compound I of the present invention for treating and / or preventing a thromboembolic disease. A method for treating a disease is provided. In another aspect of the present invention, the present invention provides a method for inhibiting blood coagulation, comprising administering to a mammal in need of treatment an effective amount of the compound I of the present invention in inhibiting blood coagulation. The treatment of thrombin inhibition, blood coagulation inhibition and thromboembolic disorders contemplated by the methods of the present invention suitably include both medical treatment and / or prevention.

In a further embodiment of the present invention, the present invention provides:
It relates to the treatment of physical conditions in which inhibition of thrombin is required in a human or animal. The compounds of the invention are useful in treating or preventing thrombosis in animals, including humans, and hypercoagulability in blood and tissues. The compounds are highly effective in preventing thrombosis and hypercoagulability in blood and tissues. The compounds are highly effective in treating and / or preventing venous thrombosis and pulmonary embolism, myocardial ischemia, heart failure, unstable angina, stroke caused by thrombosis and peripheral arterial thrombosis. further,
The compounds are useful in treating or preventing atherosclerotic diseases such as coronary artery disease, cerebral artery disease and peripheral artery disease. Further, the compounds can be used with thrombolytics in heart failure. In addition, the compounds are useful for thrombosis, percutaneous transluminal angioplasty (PT
CA) and for the prevention of reocclusion after coronary artery bypass graft surgery. Further, the compounds are useful for preventing post-microsurgical rethrombosis. In addition, the compounds are useful for anticoagulant therapy associated with artificial organs and heart valves. In addition, the compounds are useful for anticoagulant therapy in hemodialysis and sporadic intravascular coagulation. In addition, the compounds are useful in cleaning catheters and medical devices used in patients and as anticoagulants in preserving blood, plasma and other blood products. In addition, the compounds are useful in other diseases where blood coagulation is a root cause process or secondary pathogen, such as cancer metastasis, inflammatory diseases such as arthritis, and diabetes.
The anticoagulant compound of the present invention can be orally, intravenously (iv), intramuscularly (im) or subcutaneously (sc) or the like.
It is administered parenterally.

The specific dose of the compound of the present invention for obtaining an effective therapeutic and / or prophylactic effect, of course, encompasses each case, such as the compound to be administered, the frequency and interval of administration, the route of administration and the subject to be treated. Determined according to the environment. A typical daily dose for each of the above uses is from about 0.01 mg / kg to about 1000 mg / kg.
It is. The dosage regimen may be, for example, in prophylactic use,
It may vary depending on whether it is administered once a day or multiple times three or five times a day. In the context of treating severe conditions, the compounds of the present invention may be administered by intravenous infusion from about 0.01 mg / kg / hour to about 20 mg / kg / hour, preferably about 0.1 mg / kg / hour.
Administer at a rate of g / hr to about 5 mg / kg / hr. The method of the present invention relates to a tissue plasminogen activator (tP
A), modified t-PA, streptokinase or urokinase may be combined with a clot dissolving agent.
When clot formation occurs and the artery or vein is partially or completely blocked, a clot lysing agent is typically used.
The compound of the present invention can be administered before or simultaneously with the administration of the solubilizing agent, or after the use of the solubilizing agent, and is preferably administered simultaneously with aspirin to prevent re-occurrence of clot formation. The method of the present invention provides a platelet glycoprotein receptor (IIb) that inhibits platelet aggregation.
/ IIIa) may be performed in combination with an antagonist. The compounds of the present invention can be administered prior to or simultaneously with the administration of IIb / IIIa, or after its use, in order to prevent re-occurrence of clot formation. The method of the present invention may be performed in combination with aspirin. The compounds of the present invention can be administered prior to or simultaneously with the administration of aspirin, or after its use, to prevent re-occurrence of clot formation. As mentioned above,
Preferably, the compounds of the present invention are administered in combination with a clot dissolving agent and aspirin.

The present invention also provides a pharmaceutical composition used for the above-mentioned treatment method. The pharmaceutical composition of the present invention comprises a thrombin inhibiting effective amount of Compound I and a pharmaceutically acceptable carrier,
Contains excipients or diluents. For oral administration, the antithrombin compound of the present invention is formulated in the form of gelatin capsules or tablets containing excipients such as binders, lubricants, and disintegrants. For parenteral administration, physiological saline (0.
9%), 5% dextrose, Ringer's solution (0.9%)
The antithrombin is formulated in a dosage form diluted with a pharmaceutically acceptable diluent such as. The compounds of the present invention can be formulated as a formulation containing from about 0.1 mg to about 1000 mg of the compound per unit dose. Preferred are compounds of the present invention in the form of a pharmaceutically acceptable salt, such as a sulfate, acetate or phosphate. For example, a unit dosage form containing 5 mg of a compound of the present invention as a pharmaceutically acceptable salt in a 10 ml sterile glass ampoule. Other unit dosage forms include about 10 mg of a compound of the present invention as a pharmaceutically acceptable salt in a sterile glass ampoule containing 20 ml of isotonic saline.

The compound of the present invention can be administered by various routes such as oral, enteral, transdermal, subcutaneous, intravenous, intramuscular, and nasal. The compound of the present invention is preferably formulated immediately before administration. Another embodiment of the present invention is a pharmaceutical composition comprising an effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient. . The active ingredient is contained in such a preparation in an amount of 0.1 to 99.9% by weight of the preparation. "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other formulation ingredients and must not be harmful to the recipient. means. The pharmaceutical compositions of the present invention are manufactured using known procedures and readily available ingredients. The compound of the present invention can be formulated as an immediate-release, sustained-release or slow-release preparation of the active ingredient using known procedures. In the production of the composition of the present invention, the active ingredient is usually mixed with a carrier, diluted with a carrier, or a capsule,
Enclosed in a carrier, which may be in the form of a sashimi, paper, or other container. When the carrier acts as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions may be tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid vehicles), or soft and hard gelatin capsules, suppositories. , Sterile injectable solutions and sterile encapsulated powders.

The following formulation examples are intended only for a detailed explanation and do not limit the scope of the present invention.
The “active ingredient” is, of course, the compound I of the present invention or a pharmaceutically acceptable salt or solvate thereof. Formulation Example 1 A hard gelatin capsule is prepared using the following components. Amount mg / capsule Active ingredient 250 Starch, dried 200 Magnesium stearate 10 Total 460mg

Formulation Example 2 A tablet is prepared using the following components. Amount mg / tablet Active ingredient 250 Cellulose, microcrystal 400 Silicon dioxide (melted) 10 Stearic acid 5 Total 665 mg The components are mixed and each is tabletted into tablets weighing 665 mg.

Formulation Example 3 An aerosol solution is prepared using the following components. Weight Active Ingredient 0.25 Ethanol 25.75 Propellant 22 (Chlorodifluoromethane) 74.00 Total 100.00mg Mix Active Ingredient with Ethanol and Mix Propellant
In addition to part of 22, cool to -30 ° C and transfer to filling equipment. The required amount is then placed in a stainless steel container and the propellant
Dilute with the rest of 22. Attach a set of valves to the container.

Formulation Example 4 A tablet containing 60 mg of the active ingredient is prepared as follows. Active ingredient 60 mg Starch 45 mg Microcrystalline cellulose 35 mg Polyvinylpyrrolidone (as a 10% solution in water) 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg Total 150 mg Active ingredient, starch and cellulose No. 45 Mesh
Pass through US sieve and mix thoroughly. Mix the aqueous solution containing polyvinylpyrrolidone with the remaining powder, and mix the mixture.
Pass through No. 14 mesh US sieve. The granules thus produced are dried at 50 ° C. and passed through a No. 18 mesh US sieve. Add sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh US sieve, to the granules, mix them, and tablet each with a tablet machine into tablets weighing 150 mg. .

Formulation Example 5 Capsules each containing 80 mg of the active ingredient are prepared as follows. Active ingredient 80 mg starch
59 mg microcrystalline cellulose
59 mg Magnesium stearate 2 mg Total 200 mg The active ingredient, cellulose, starch and magnesium stearate are mixed, passed through a No. 45 mesh US sieve, and filled into hard gelatin capsules of 200 mg amount.

Formulation Example 6 A suppository containing 225 mg of the active ingredient is prepared as follows. Active ingredient 225 mg Saturated fatty acid glyceride 2,000 mg Total 2,225 mg Pass the active ingredient through No.60 mesh US sieve,
Suspend in saturated fatty acid glycerides that have been melted using minimal heat. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.

Formulation Example 7 A suppository containing 50 mg of the active ingredient per 5 ml dose is prepared as follows. Active ingredient 50 mg Sodium carboxymethylcellulose 50 mg Syrup 1.25 ml Benzoic acid solution 0.10 ml Flavoring agent qv Coloring agent qv Add distilled water to make a total of 5 ml. Pass the active ingredient through No. 45 mesh US sieve and mix with sodium carboxymethylcellulose and syrup to make a smooth paste. The benzoic acid solution, flavor and color are diluted with some of the water and added, with stirring. Add enough water,
Make the capacity necessary.

Formulation Example 8 A formulation for intravenous administration is prepared as follows. Active ingredient 100 mg Isotonic solution 1,000 ml In general, a solution of the above ingredients is intravenously administered to a subject at a rate of 1 ml per minute.

The ability of the compounds of the present invention to be effective orally active thrombin inhibitors is evaluated. Compound I provided by the present invention selectively inhibits thrombin in mammals. Thrombin inhibition is determined by the fact that thrombin is a chromogenic substrate, N-benzoyl-L-phenylalanyl-
L-valyl-L-arginyl-p-nitroanilide (in another expression, N-benzoyl-L-Phe-L-Val
-L-Arg-p-nitroanilide) as measured by an in vitro inhibition of thrombin amidase activity as determined by an assay that hydrolyzes it. The assay was performed in buffer (0.03 M Tris, 0.15 M NaC).
1, pH 7.4) and 25 μl of a human thrombin solution (purified human thrombin, Enzyme Research Laboratories, South Bend, Indiana, 8 NIH units ml) and a solvent (50% aqueous methanol,
v: Perform by mixing 25 μl of a solution of the test compound in v). Then, an aqueous solution of the chromogenic substrate (0.25 mg /
ml) and the rate of hydrolysis of the substrate is measured by monitoring the response at 405 nm to the release of p-nitroanilide. A standard curve is constructed by plotting the free thrombin concentration against the rate of hydrolysis. The hydrolysis rate obtained with the test compound is then converted to the "free thrombin" value in each assay using a standard curve. Bound (bound test compound) thrombin is calculated by subtracting the amount of free thrombin observed in each assay from the known initial amount used in the assay. The amount of free inhibitor in each assay is calculated by subtracting the number of moles of bound thrombin from the number of moles of added inhibitor (test compound).

The Kass value is a tentative equilibrium constant in the reaction between thrombin and test compound I. Thrombin + I⇔ Thrombin-I Kass = [Thrombin-I] / [(Thrombin) ×
(I)] Kass is calculated in the concentration range of the test compound, and the average value is reported in units of liter / mol. Generally, the Kass value of the thrombin inhibitory compound I of the present invention is 0.1 × 10 ⁶ liter / mol or more. For example, each of the particularly preferred compounds of the invention described above had a Kass value of at least 5 × 10 ⁶ L / mol. For example,
The Kass values of the compounds of Examples 7 and 8 were 57
× 10 ⁶ L / mol and 80 × 10 ⁶ L / mol.

Substantially in accordance with the procedure described above for human thrombin, using other human blood coagulation serine proteases and fibrinolytic serine proteases, in a suitable chromogenic substrate described below, the coagulation factor serine protease and fibrin The selectivity of the compound of the present invention with respect to the lytic serine protease and the fact that the compound of the present invention does not substantially interfere with human coagulated plasma fibrin lysis are evaluated. Human factors X, Xa, IXa, XIa and XII
a: Enzyme Research Laboratories, South
Bend, Indiana; human urokinase is Leo
Purchased from Pharmaceuticals, Denmark and recombinant activated protein C (aPC) is prepared at Eli Lilly and Company substantially according to US Pat. No. 4,981,952. Chromogenic substrate: N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa); N-Cbz-D-Arg-G
ly-Arg-p-nitroanilide (Factor Xa substrate for Factor IXa assay); Pyroglutamyl-Pro-
Arg-p-nitroanilide (factor XIa and aPC
HD-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); and pyroglutamyl-G
ly-Arg-p-nitroanilide (for urokinase)
Is purchased from Kavi Witrum, Stockholm, Sweden or Midwest Biotech, Fishers, Indiana. Bovine trypsin is purchased from Worthington Biochemicals, Freehold, NJ, and human plasma kallikrein is purchased from Mold Witzram, Stockholm, Sweden. Chromogenic substrate H-Pro- for plasma kallikrein
Phe-Arg-p-nitroanilide is purchased from Mold Vitrum, Stockholm, Sweden. Substrate N-benzoyl-P for human thrombin and trypsin
He-Val-Arg-p-nitroanilide may be synthesized from a commercially available reaction product using a known peptide coupling method according to the above-mentioned procedure for producing the compound of the present invention, or may be synthesized from Midwest Biotech, Fishers. ， Purchase from Indiana.

Human plasma is purchased from Boehringer Mannheim, Indianapolis, Indiana;
PA is purchased from American Diagnostica, Greenwich, Connecticut as a single-stranded activity control;
Modified-t-PA6 (mt-PA6) is prepared by Eli Lilly and Company by known procedures (see Burke et al., J. Biol, Chem., 265, 5120-5177 (1990)). Plasmin chromogenic substrate HD-Val-Leu-
Lys-p-nitroanilide and tissue plasminogen activator (t-PA) substrate HD-Ile-P
ro-Arg-p-nitroanilide is purchased from Mold Vitrum, Stockholm, Sweden. The three-letter symbols Ile, Glu,
Pro, Arg, Phe, Val, Leu and Lys
Are the corresponding amino acids, namely isoleucine, glutamic acid, glycine, proline, arginine,
Represents phenylalanine, valine, leucine and lysine. Preferably, the thrombin inhibitor does not interfere with fibrinolysis induced by urokinase, tissue plasminogen activator (t-PA) and streptokinase. This means that streptokinase,
It is important when the inhibitor is used for therapy in conjunction with thrombolysis treatment using t-PA or urokinase, or when the inhibitor is used as an endogenous fibrinolysis-tolerant (for t-PA or urokinase) antithrombin agent. . In addition to being non-interfering with the amidase activity of fibrinolytic proteases, such fibrinolytic system acceptability is exploited by human clotting plasma and its breakdown by the respective fibrinolytic plasminogen activator. You can experiment.

Materials Anesthetized mixed breeding hound (Hampelton-LR amphoteric)
E, Kalamazoo, Michigan, USA) obtain dog plasma by venipuncture into 3.8% citrate. According to the method described above, fibrinogen is prepared from fresh dog plasma, and human fibrinogen is prepared from fraction I-2 of ACD human blood collected on the day. Smith, Biochem. J., 185, 1-11 (1980); and Smith, et al., Bi.
ochemistry, 11, 2958-2967 (1972). Human fibrinogen (98% pure / no plasmin) is obtained from American Diagnostica, Greenwich, Connecticut. Radiolabeling of the fibrinogen I-2 preparation is performed as described above. Smith et al. Biochemistry, 11, 2958-2967
(1972). Purchase Leuro Pharmaceuticals, Denmark, Urokinase for 2200 Proog units / vial. Streptokinase is purchased from Hoechst-Roussel Pharmaceuticals, Somerville, NJ.

Method-Lysis of Human Coagulated Plasma with t-PA
Effect of 100 μl of human plasma containing 0.0229 μCi of 125-I labeled fibrinogen is added to 50 μl of thrombin to form human coagulated plasma in a microtube. Urokinase or streptokinase (5
The experiment for coagulated plasma lysis is performed by adding 50 μl (0, 100 or 1000 units / ml) and incubating at room temperature for 20 hours. After the incubation, the tubes are centrifuged in a Beckman centrifuge.
25 μl of the supernatant was added to 0.03 M Tris / 0.15 M NaC
Perform gamma counting in 1.0 ml of 1 buffer. 100% lysis, a count control, is obtained by removing thrombin (replacement with buffer). Concentration 1,
Thrombin inhibitors are evaluated for possible interference with fibrinolysis by including the inhibitor compound in the loading solution added at 5 and 10 μl / ml. By linear extrapolation from the data points to a value representing 50% dissolution, at the specified concentration of fibrinolytic agent,
Calculate the approximate IC ₅₀ value.

Anticoagulant Active Material Anesthetized Mixed Breeding Hound (Hampelton-LR amphoteric)
E, Kalamazoo, Michigan, USA) or anesthetized male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, Indiana, USA) to 3.8% citrate. Dog and rat plasma are purchased by puncture. AC collected on the day according to the method described above
D Human fibrinogen is prepared from fraction I-2 of human blood. Smith's Biochem. J., 185, 1-11 (1980);
And Smith et al., Biochemistry, 11, 2958-2967 (1972).
Human fibrinogen (purity 98% / without plasmin)
Is also purchased from American Diagnostics, Greenwich, Connecticut. Coagulation reagents ACTIN, thromboplastin and human plasma are purchased from Baxter Healthcare Corporation, Dade Division, Miami, Florida. Anticoagulation assays in plasma are performed using bovine thrombin purchased from Parque-Davis (Detroit, MI).

Method Measurement of Anticoagulation The anticoagulation assay procedure is as described above. Smith et al.
Thrombosis Research, 50, 163-174 (1988). All coagulation assay measurements are performed using a CoA screener coagulator (American Labor, Inc.). To 0.05 ml of test plasma, 0.05 m of physiological saline
l and thrombin 0.05 ml (10 NIH / ml)
The thrombin time (TT) is measured by adding Test plasma 0.05 ml with actin reagent 0.05 m
120 seconds with CaCl ₂ (0.02
The activated partial thromboplastin time (APTT) is determined by incubating with M). Prothrombin time (PT) is measured by adding 0.05 ml of saline and 0.05 ml of thromboplastin-C reagent to 0.05 ml of test plasma. To human or animal plasma, compound I at a wide range of concentrations
The time prolongation effect in the T, APTT and PT assays is measured. In each assay, a linear extrapolation is performed to assess the concentration required to double the clotting time. 100 ng of each of the above-listed particularly preferred examples of the present invention.
/ Ml was determined to have a TT value of less than or equal to. For example, the TT, APTT, and PT values of the compound of Example 7 were 30, 280, and 430, and the compounds of Example 8 were 20, 170, and 380.

Experimental animals Male Sprague-Dawley rats (350-425 g,
Harlan Sprague-Dowry Incorporated, Indianapolis, Indiana, USA
Is anesthetized with xylazine (20 mg / kg, sc) and ketamine (120 mg / kg, sc) and kept on a heated water blanket. Insert a cannula for jugular infusion. Arteriovenous shunt model A 20 cm long tube of polyethylene PE60 is cannulated into the left jugular vein and right carotid artery. A 6 cm center of a larger tube (PE190) with cotton thread (5 cm) in the lumen is friction fit between the longer sections to complete the arteriovenous shunt circuit. After circulating blood through the shunt for 15 minutes, the cotton thread is carefully removed and weighed. The weight of the wet thread is subtracted from the total weight of the thread and blood vessels [JR Smith, Br J Pharmacol, 77: 2.
9, 1982].

FeCl ₃ Model of Arterial Injury The carotid artery is isolated by midline ventral cervical incision. A thermocouple is placed under each artery and the temperature of the container is continuously recorded with a strip chart recorder. A vertically cut tube (0.0
A cuff of 58 ID x 0.077 OD x 4 mm, Baxter medical grade silicone) is placed directly over the thermocouple around each artery. FeCl ₃ .6 hydrate is dissolved in water, and the concentration (20%) is expressed in terms of the actual weight of FeCl ₃ alone.
To damage the artery and induce a thrombus, pipette 2.85 μl into the cuff and dip the artery over a thermocouple probe. Arterial occlusion is indicated by a rapid decrease in temperature. Time to occlusion is reported in minutes,
The elapsed time between the application of FeCl ₃ and the rapid drop in vessel temperature is displayed [KD Kurtz, Thromb. Res., 60: 269,199.
See 0].

Spontaneous thrombolysis model In vitro data suggest that peptide thrombin inhibitors inhibit thrombin and, at high concentrations, other serine proteases such as plasmin and tissue plasminogen activator. To determine whether compounds of the invention inhibit fibrinolysis in vivo, the rate of spontaneous thrombolysis is determined by injecting labeled coagulated whole blood into the pulmonary circulation. Rat blood (1 ml) was added to bovine thrombin (4I
U, Parque Davis) and ¹²⁵ I human fibrogen (5 μCi, ICN), quickly transfer to a silicon tube and incubate at 37 ° C. for 1 hour.
The grown thrombus is peeled from the tube, cut into 1 cm segments, washed three times with normal saline, and each segment is counted on a gamma counter. A segment of known count is aspirated into a catheter and then injected into the jugular vein. A catheter tip is introduced in the vicinity of the right blood vessel, driving out blood clots and floating in the pulmonary circulation.
One hour after injection, hearts and lungs are collected and counted separately. Thrombi are expressed as a percentage calculated from the following formula:% thrombus = [(infused cpm-lung cpm) / infused cpm] x 100 The fibrinolysis of the infused clot occurs in a time-dependent manner. (JP Krozel's Cardiovas. Pharmacol., 12: 520,
1988).

Coagulation parameters Plasma thrombin time (TT) and activated partial thromboplastin time (APTT) are measured with a fibrometer. A blood sample is collected via a jugular vein catheter,
Aspirate into syringe containing sodium citrate (3.8%, 1-9 parts of blood). To determine TT, rat plasma (0.1 ml) was mixed with saline (0.1 ml) and bovine thrombin (0.1 ml, 30 U / m in Tris buffer).
l; Parque Davis) at 37 ° C.
To measure APTT, plasma (0.1 ml) and AP
The TT solution (0.1 ml, Organon Technica) was incubated for 5 minutes, and CaCl ₂ (0.1 ml,
0.025M). The assay is performed twice, and the average value is determined.

Indicator of bioavailability Measuring biological activity, ie, plasma thrombin time (TT), is based on the inhibition of thrombin by the parent compound alone.
An alternative to the parent compound assay, based on the assumption that T is increased. Intravenous (i.
v) The time course of the effect of the thrombin inhibitor on TT is measured after bolus administration and after oral (po) administration to anaesthetized fasted rats. Two populations of rats are used due to limited blood volume and the number of data points required to measure the time course from the time of treatment to when the response returns to pre-treatment values. For each sample population, another continuous time point is displayed. Using the average TT over time, the area under the blood concentration-time curve (AU
Calculate C). An index of bioavailability is calculated from the following formula and expressed as a relative activity percentage.
The AUC of the plasma TT time course is determined and adapted for dose. This measure of bioavailability is referred to as "percent relative activity" and is calculated from the following formula:% relative activity = AUCpo / AUCiv x dose iv / dose
po × 100

Compounds Solutions of compounds were prepared fresh daily as saline solutions and bolus injections or perfusion started 15 minutes prior to the experiment (15 minutes in the arteriovenous shunt model, 15 minutes in FeCl ₍ 60 minutes for ₃ models and spontaneous thrombolysis model). The bolus injection volume is 1 mg / kg for iv, 5 mg / kg for po, and the perfusion rate is 3 ml / hour.

Statistical considerations The results are expressed as mean ± SEM. One-way analysis of variance is used to detect statistically significant differences and Dunnett's test is applied to determine that the means are different. The significance level of rejection of the null hypothesis of equal mean is P <0.05.

Experimental animal Male dog (beagle; age: 18 months to 2 years; weight 12)
~ 13kg; Marshall Farm, North Rose,
New York 14516) was fasted overnight and administered 240
Give the Purina Guaranteed Prescription Diet (Purina Mills, St. Louis, MO) a minute before. Water is to be drunk at any time. Maintain room temperature 66-74 ° F., relative humidity 45-50% and illuminate from 6:00 to 18:00. The pharmacokinetic model test compound is dissolved in sterile 0.9% physiological saline at a concentration of 5 mg / ml and compounded immediately before administration. Dogs are dosed orally with test compound at a dose of 2 mg / kg in a single dose. 0.25, 0.5, 0.75, 1, 2, 3,
At 4 and 6 hours, a blood sample (4.5 ml) is taken from the head vein. The sample is placed in a vacutainer tube containing citrate and stored on ice until plasma is removed by centrifugation. Analyze the plasma sample by HPLC MS. The plasma concentration of the test compound is recorded and used to determine the pharmacokinetic parameters: elimination rate constant, Ke; total clearance, Clt;
V _D ; time of maximum concentration of test compound in plasma, Tmax; T
max concentration of test compound, max; plasma half-life; t _0.5 ; and area under the blood concentration-time curve, AU
C: Fraction of absorbed test compound, F, is calculated.

Surgical preparation and instrumentation of a canine model dog with coronary thrombosis is described by Jackson et al., Circulation, 82, 830-940 (1).
990). Mixed breeding hounds (6-7 months old, amphoteric, Hazelton-LRE, Kalamazoo, MI,
USA) with sodium pentobarbital (30
mg / kg, intravenous administration), keep warm, and ventilate room air. PO ₂ and PCO _{2 in} blood by adjusting respiratory volume and respiratory rate
And maintain the pH within the normal range. Insert a hypodermic needle electrode and record the Lead II ECG. The left jugular vein and carotid artery are isolated by left medial-lateral neck incision. Arterial blood pressure (AB
P) pre-adjusted mirror converter (Model MPC-50)
0, Miller Instruments, Houston, TX,
(USA). Sample blood during the experiment through a cannula into the jugular vein. In addition, the test compound is administered via a cannula through the femoral vein of both hind paws. A left thoracotomy is performed in the fifth intercostal space and the heart is suspended from the pericardial cradle. 1-2 of the left circumflex coronary artery (LCX)
A cm segment is isolated closer to the first main ventricular branch. A wire anode (Teflon-coated, 30-gauge silver-plated copper wire) with a 26-gauge needle at the end having a length of 3 to 4 mm is inserted into the LCX and placed in contact with the intimal surface of the artery (at the end of the experiment). Confirm). The stimulation circuit is completed by placing the cathode at the subcutaneous site.
An adjustable plastic occluder is placed over the electrode area around the LCX. Pre-adjusted electromagnetic flow probe to measure coronary artery blood flow (CBF) (Carolina Medical Electronics, King, NC, USA)
Is placed around the LCX near the anode. The occluder is adjusted so that the inhibition of the hyperemic blood flow response observed after a 10 second mechanical occlusion of the LCX is 40-50%. All hemodynamic and ECG measurements are recorded and analyzed on a data acquisition system (Model 3000, Modular Instruments, Malvern, PA, USA).

Thrombus Formation and Compound Dosing Formulation Electrolytic trauma to the intima of the LCX is created by applying a direct current (DC) of 100 μA to the anode. The electric current is applied for 60 minutes, and then the energization is stopped whether or not the blood vessel is occluded. Thrombus formation progresses spontaneously until the LCX is completely occluded (measured as an increase in the zero CBF and ST segment). Compound administration begins one hour after the formation of the embolic thrombus.
The injection of the compound of the present invention is performed using a thrombolytic agent (tissue plasminogen activator, streptokinase, APSAC).
0.5) and 1 mg / k
Perform for 2 hours at a dose of g / hour. Reperfusion is performed 3 hours after administration of the test compound. Reocclusion of the coronary arteries after successful thrombolysis is defined as zero CBF lasting more than 30 minutes.

Hematological considerations and template bleeding time
Measurement hematology analyzer (Cell-Dyn900,
Sequoia Turner, Mount View, CA, US
Using A), the total blood cell count, hemoglobin, and hematocrit are measured from a 40 μl sample of citric acid (3.8%)-treated blood (1 part of citrate: 9 parts of blood). With a Simplate II bleeding time device (Organon Technica Durham, NC, USA)
Measure gingival template bleeding time. The device is used to make horizontal incisions in the upper and lower gingiva of the left jaw of the dog. Each cut is 3 mm wide and 2 mm deep. The incision is made and the time at which bleeding begins is measured using a stopwatch. Use cotton cotton to absorb blood from the cut. The template bleeding time is the time from incision to cessation of bleeding. Immediately before administration of the test compound (0 min),
Bleeding time is measured 60 minutes after injection, at the end of test compound administration (120 minutes) and at the end of the experiment. All data are analyzed by a one-way analysis of variance (ANOVA) followed by a Student-Neuman-Keuls post hot t test to determine the level of significance. Repeated measures ANOVA are used to determine significant differences between time points during the experiment. Values are determined to be statistically different at least at the level of p <0.05. All values are means ± SEM. All experiments are performed according to the American Physiological Society's guidance policy. Further details regarding the procedure are given in Jackson et al., J. Cardiovsc. Pharmacl., 21, 587-599 (1993).

The following examples are provided to further illustrate the present invention and do not limit the invention. The meanings of the abbreviations used in the examples are as follows. Amino acid: Azt = acetidine-2-carboxylic acid, Ph
e = phenylalanine, hPro = homo-proline, P
ro = proline, Cha = β-cyclohexylalanine, Ohi = [2S- (2α, 3aβ, 7aβ)]-octahydro-indole-2-carboxylic acid, 3-Pig
= D-cis [4aR, 8aR] -3-perhydroisoquinolinecarboxylate Boc = t-butyloxycarbonyl Bn = benzyl BOP-Cl = bis (2-oxo-3-oxazolidinyl) phosphinic acid chloride t-Bu = t -Butyl n-BuLi = butyllithium 18-crown-6 = 1,4,7,10,13,16-
Hexaoxacyclooctadecane DIBAL = diisobutylaluminum hydride DMF = dimethylformamide DMSO = dimethylsulfoxide Et = ethyl EtOAc = ethyl acetate Et ₂ O = diethyl ether EtOH = ethanol FAB-MS = fast atom bombardment mass spectrum FD-MS = field Di Soap HPLC = high performance liquid chromatography HRMS = high resolution mass spectrum HOBT = 1-hydroxybenzotriazole hydride i-PrOH = isopropanol IR = infrared spectrum Me = methyl MeOH = methanol NMR = nuclear magnetic resonance RPHPLC = reversed phase height Performance liquid chromatography SiO ₂ = silica gel TEA = triethylamine THF = tetrahydrofuran TLC = thin layer chromatography Matography Ts = Tosyl (p-toluenesulfonyl) The following parameters were used in the preparative RPHLC: Solvent A: 0.05% aqueous hydrochloric acid (1.5 ml of concentrated hydrochloric acid dissolved in 3 L of water); Solvent B: Acetonitrile; Gradient: as defined in each example; Column: Vdac C
₁₈ -5 cm × 25 cm; flow rate: 10 ml / min. Unless otherwise stated, adjustment and setting of pH was performed with acid or base solutions. ¹ H-NMR indicates that a sufficient NMR spectrum has been obtained for the described compound. IR
Indicates that sufficient infrared spectra were obtained for the described compounds.

Example 1 D-Cha-Pro-4- (NHCH ₂ -trans-CHC
H) Imidazole.2HCl (D-cyclohexylalanyl-N-[(E) -3- (imidazol-4-yl)
Prop-2-enyl] -L-prolinamide dihydrochloride)

Embedded image D-Cha-Pro-4- ( NHCH 2 - trans -CHC
_{H) C 3 H 3 N 2} · 2HCl A) Boc-D-Cha-Pro-OH in dichloromethane (360mL) Boc-D-Cha -O
A solution of H (50.4 g, 185 mmol) was cooled to 0 ° C. and then N-hydroxysuccinimide (22.3
g, 194 mmol). Then, 1,3-dicyclohexylcarbodiimide (39.0 g, 189 mmol) was added in two portions as a solution in dichloromethane (90 mL). After stirring at 0 ° C for 3 hours, L-Pro-OH
(27.6 g, 240 mmol) and N, N-diisopropylethylamine (30.9 g, 239 mmol) were added. After stirring for a further 3 hours between 0 ° C. and 10 ° C., the mixture was filtered over diatomaceous earth. The filter cake was rinsed with dichloromethane (100 mL); the filtrates were then combined and concentrated in vacuo. The residual oil was partitioned between ethyl acetate (100mL) and 0.625M aqueous sodium bicarbonate (320mL). The layers were separated and the organic phase was washed with 0.625M aqueous NaHCO ₃ (80 mL). The bicarbonate extracts were then combined and washed with ethyl acetate (100 mL). The aqueous phase was then stirred with ethyl acetate (300 mL) and then acidified with 12N HCl (about 37 mL). The layers were separated and the acidic aqueous phase was extracted with ethyl acetate (100mL). The ethyl acetate extracts were combined and concentrated under reduced pressure. The residue was slurried with a minimum amount of ethyl acetate, filtered, washed again with ethyl acetate, and then dried to 50.1 g (73
%) As a white powder. ¹ H-NMR, FAB-MS m / e 369.2 (M
H ⁺⁾ as the element analysis _{(C 19 H 32 N 2 O} 5): Calculated: C, 61.93; H, 8.75 ; N, 7.60 Found: C, 62.01; H, 8 . 96; N, 7.75

B) Methylurocanate.HCl Anhydrous hydrochloric acid was bubbled into a suspension of urocanic acid (69 g, 500 mmol) in methanol (1 L) until the solution was saturated. The solution was then heated to reflux while stirring, the next morning the solution was cooled and the solvent was removed under reduced pressure. The residue was washed twice with diethyl ether, filtered and then dried under reduced pressure to give 93 g (98%) of a white solid. IR ¹ H-NMR FD-MS, m / e 152.0 (M ⁺ ) Elemental analysis (as C ₇ H ₈ N ₂ O ₂ .HCl): Calculated: C, 44.58; H, 4.81; N, 14.85 Found: C, 44.46; H, 4.82; N, 14.78.

C) Methyl N-Ts-urocanate A stirred suspension of methylurocanate.HCl (20 g, 106 mmol) in dichloromethane (300 mL) was added.
-Toluenesulfonyl chloride (21.2 g, 111 mmol) followed by triethylamine (24.4 mL, 1
80 mmol) was added. After 16 hours, the solvent was removed under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic phase was washed twice with saturated NH ₄ Cl and twice with saturated NaHCO ₃ , then dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with 5% ethyl acetate / chloroform; the product-containing fractions were then combined,
Concentration under reduced pressure gave 20.4 g (63%) of a white solid. IR ¹ H-NMR FD-MS, m / e 306 (M ⁺ ) Elemental analysis (as C ₁₄ H ₁₄ N ₂ O ₄ S.HCl): Calculated: C, 54.89; H, 4.61; N , 9.14 found: C, 55.17; H, 4.64; N, 9.02.

D) 4- (HOCH ₂ -trans-CHC
H) -1-Ts-imidazole At 0 ° C., a stirred solution of methyl N-Ts-urocanate (10 g, 33 mmol) in THF (250 mL) was added with DI.
BAL (1 M toluene solution, 65 mL, 65 mmol)
Was added. After 2 hours, the solution was diluted with ethyl acetate (500 mL) and stirred vigorously with aqueous potassium sodium tartrate solution. After 30 minutes, the layers separated, the organic layer was washed with brine, dried over Na ₂ SO _4, filtered, and concentrated under reduced pressure to give a white solid 8.6g (95%). IR ¹ H-NMR FD-MS, m / e 278 (M ⁺ ) Elemental analysis (as C ₁₃ H ₁₄ N ₂ O ₃ S): Calculated: C, 56.10; H, 5.07; N, 10 .06 found: C, 56.40; H, 5.21; N, 9.86.

E) 4- (BrCH ₂ -trans-CHC
H) -1-Ts-imidazole 4- (HOCH ₂ -trans- in THF (250 mL)
A stirred solution of CHCH) -1-Ts-imidazole (7.0 g, 25 mmol) was added to carbon tetrabromide (12.5 g, 38 mmol) followed by triphenylphosphine (9.9 g, 3 mmol).
8 mmol) was added. After stirring for 2 hours, the solvent was removed under reduced pressure, then the residue was dissolved in chloroform,
Chromatography on a column packed dry with silica gel, eluting with a step gradient of 0-50% ethyl acetate / hexane. The product containing fractions were combined and concentrated under reduced pressure to give 7.8 g (61%) of a white solid. IR ¹ H-NMR FD-MS, m / e 342 (M ⁺ ) Elemental analysis (as C ₁₃ H ₁₃ BrN ₂ O ₂ S): Calculated: C, 45.76; H, 3.84; N, 8 .21 found: C, 46.03; H, 3.97; N, 8.25.

F) 4- (Boc ₂ NCH ₂ -trans-C
NaH (60% dispersed oil in HCH) -1-Ts-imidazole THF (150 mL)
(0.7 g, 17.6 mmol) in THF.
A solution of di-tert-butyl iminodicarboxylate (3.82 g, 17.6 mmol) in (25 mL) was added slowly, followed by 4- (BrC in THF (25 mL).
H ₂ - was added a solution of trans -CHCH) -1-Ts- imidazole (4.0 g, 11.7 mmol). 20
After stirring for an hour, the solvent was removed under reduced pressure, and the residue was partitioned between ethyl acetate and water. The ethyl acetate phase was washed with saturated aqueous NH ₄ Cl, saturated aqueous NaHCO ₃ and brine, then dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was chromatographed on silica gel eluting with a step gradient of 10-50% ethyl acetate / hexane and the product containing fractions were combined and concentrated in vacuo to give 3.3 g (59%) of a white solid. Obtained. IR ¹ H-NMR FD-MS, m / e 477 (M ⁺ ) Elemental analysis (as C ₂₃ H ₃₁ N ₃ O ₆ S): Calculated: C, 57.85; H, 6.54; N, 8 .80 found: C, 58.08; H, 6.55; N, 8.84.

G) 4- (H ₂ NCH ₂ -trans-CHC
H) -1-Ts-imidazole.HCl 0 ° C., methyl 4- (25 mL) in dichloromethane (25 mL).
To a stirred solution of (Boc ₂ NCH ₂ -trans-CHCH) -1-Ts-imidazole (4.74 g, 9.9 mmol) was added trifluoroacetic acid (25 mL). After stirring for 1.5 hours, the solvent was removed under reduced pressure, and the residue was suspended in diethyl ether with vigorous stirring. The solid was filtered, washed again with diethyl ether, filtered, and dried under reduced pressure to give 3.7 g (95%) of the TFA salt as a white solid. Part of TFA salt (500m
g) was dissolved in water (50 mL) and 1N HCl (5 mL), washed with ethyl acetate, concentrated under reduced pressure to 20 mL, then lyophilized to give the title hydrochloride. IR ¹ H-NMR FD-MS, m / e 278.1 (MH ⁺ )

H) Boc-D-Cha-Pro-NH-4-
(CH ₂ -trans-CHCH) -1-Ts-imidazole In flask 1 at 0 ° C., 4- (H ₂
NCH ₂ - trans -CHCH) -1-Ts- imidazo -
To a stirred suspension of HCI (1.3 g, 3.7 mmol) was added 4-methylmorpholine (1.13 mL, mmol). In flask 2, at −15 ° C., THF (2
Boc-D-Cha-Pro-OH (1.24 g, 5 mL).
3.4 mmol) and 4-methylmorpholine (0.38)
mL, 3.4 mmol) was added to isobutyl chloroformate (0.4).
4 mL, 3.4 mmol). After stirring for 5 minutes,
The contents of Flask 1 were added to Flask 2, and the cooling bath was removed. Next morning, the solvent was removed in vacuo and the residue was partitioned between ethyl acetate and saturated NH ₄ Cl. The organic phase was washed with brine, dried (MgS
O ₄ ), filtered and concentrated in vacuo to give the coupled product.

I) D-Cha-Pro-NH-4- (CH ₂
-Trans-CHCH) imidazole.2HCl The residue from the above step was dissolved in THF (25 mL) and HOBT (0.92 g, 6.8 mmol) was added. After stirring for 6 hours, the solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane (50 mL). TFA (50 mL) was added to the stirred solution. 1
After stirring for 2 hours, the solvent was removed under reduced pressure and the residue was partitioned between 1N HCl and ethyl acetate. The aqueous phase was washed again with diethyl ether, then with ethyl acetate, and concentrated under reduced pressure. Then the residue was washed with water (30 mL).
And then prepared by preparative RPHPLC (6
0 / 40-98 / 2 (A / B), 150 min). The product containing fractions were combined, partially concentrated, and then lyophilized to give 0.29 g (38%) of a white solid. ¹ H-NMR FAB-MS, m / e 374.3 (MH ⁺ ) Elemental analysis (as C ₂₀ H ₃₁ N ₅ O ₂ .2HCl): Calculated: C, 53.81; H, 7.45; N , 15.69 found: C, 53.58; H, 7.52; N, 15.45.

Example 2 D-Cha-Pro-NH-4- (CH ₂ CH ₂ CH ₂ ) imidazole.2HCl (D-cyclohexylalanyl-N
-[3- (Imidazol-4-yl) propyl] -L-
Production of prolinamide dihydrochloride)

Embedded image D-Cha-Pro-NH- 4- (CH 2 CH 2 CH 2) C 3 H
₃ N ₂ · 2HCl ethanol (70 mL) and D-C in water (30 mL)
ha-Pro-NH-4- ( CH 2 - trans -CHCH)
Imidazole · 2HCl (150 mg, 0.34 mmol)
To a stirred solution of was added 5% Pd / C (0.5 g). The flask was degassed and placed under a hydrogen atmosphere. After 2 hours, the solution was filtered through a pad of diatomaceous earth, and the solvent was removed in vacuo. The residue was washed with water (10m
L), filtered through a 1μ filter and then preparative RPHPLC (60 / 40-98 /
2 (A / B) for 150 minutes). The product containing fractions were combined, partially concentrated, and lyophilized to give 140 mg (92%) of a white solid. ¹ H-NMR FAB-MS, m / e 376.3 (MH ⁺ ) Elemental analysis (as C ₂₀ H ₃₃ N ₅ O ₂ .2HCl): Calculated: C, 53.57; H, 7.87; N , 15.62 Found: C, 53.30; H, 7.89; N, 15.41.

Example 3 Preparation of EtSO ₂ -D-Cha-Pro-4- (NHCH ₂ -trans-CHCH) -imidazole.HCl

Embedded image _{EtSO 2 -D-Cha-Pro-} 4- (NHCH 2 - trans _{-CHCH) C 3 H 3 N 2} · 2HCl A) EtSO 2 -D-Phe-OH THF (400mL) solution of D- phenylalanine (50
g, 300 mmol) was added N, O-bis (trimethylsilyl) -acetamide (92 g, 450 mmol). After stirring for 12 hours, the solution was added to -78.
C., and then N, N-diisopropylethylamine (58 mL, 330 mmol) was added. To this solution was slowly added sulfonylethane chloride (31 mL, 330 mL).
Mmol) and the cooling bath was removed. After stirring for 20 hours, the solvent was removed under reduced pressure, and the residue was partitioned between saturated aqueous NaHCO ₃ and ethyl acetate. The aqueous phase was washed with diethyl ether, acidified with solid citric acid and extracted twice with ethyl acetate. Combined with the ethyl acetate extract, washed with brine, dried over MgSO ₄ , filtered, and concentrated in vacuo to give 61 g (79%) of a thick, colorless, clear oil. IR ¹ H-NMR FD-MS, m / e 257 (M ⁺ ) B) EtSO ₂ -D-Phe-Pro-OBn At 0 ° C., EtSO ₂ -D-Phe-O in THF (1 L).
H (25.7 g, 100 mmol), Pro-OBn.HC
l (26.6 g, 110 mmol), HOBT (13.5
g, 100 mmol) and N, N-diisopropylethylamine (43.5 mL, 250 mL) was added to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (23 g, 120 mL).
After stirring for 20 hours, the solvent was removed in vacuo and the residue was partitioned between ethyl acetate and 1N citric acid. The organic phase was washed twice with 1N KHCO ₃ , twice with brine, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with a step gradient of 0-50% ethyl acetate / hexane. The product-containing fractions were combined, concentrated under reduced pressure,
29 g (65%) of a colorless, clear, thick oil were obtained. IR ¹ H-NMR FD-MS, m / e 444.2 (M ⁺ )

C) EtSO ₂ -D-Phe-Pro-OH EtSO ₂ -D-Phe-Pr in ethyl acetate (500 mL)
10% in a solution of o-OBn (28.5 g, 64 mmol)
Pd / C (5 g) was added. The vessel was degassed and placed under a hydrogen atmosphere. After stirring for 16 hours, the solution was filtered through diatomaceous earth, and the filter pad was washed twice with methanol and then filtered. The filtrates were combined and concentrated under reduced pressure to give 22 g (97%) of an off-white solid. IR ¹ H-NMR FD-MS, m / e 355.3 (MH ⁺ ) Elemental analysis (as C ₁₆ H ₂₂ N ₂ O ₅ S): Calculated: C, 54.22; H, 6.26; N , 7.90 found: C, 53.98; H, 6.12; N, 7.63.

D) EtSO ₂ -D-Cha-Pro-OH EtSO ₂ -D-Phe-Pr in ethanol (300 mL)
o-OH PtO ₂ (5 To a solution of (10 g, 28 mmol)
g) was added. The mixture was hydrogenated at 20 ° C. for 20 hours using a high pressure apparatus at 4.1 bar. The solution was then filtered over diatomaceous earth and concentrated to 8.1 g (80 g).
%) Of a thick oil. IR ¹ H-NMR FD-MS, m / e 361.2 (MH ⁺ )

E) EtSO ₂ -D-Cha-Pro-4- (N
HCH ₂ - By methods substantially equivalent to those described trans -CHCH) imidazole · HCl Example _{16-D, EtSO 2 -D-} Cha-Pro-4- (NHCH 2
-Trans-CHCH) imidazole.HCl to EtSO
₂ -D-Cha-Pro-OH and 4 - was prepared from (NH ₂ CH ₂ trans -CHCH) -1-Ts- imidazole · HCl. The product is prepared by preparative RPHPLC
(40 / 60-90 / 10 (A / B), 150 minutes). ¹ H-NMR FD-MS, m / e 465.1 (M ⁺ ) Elemental analysis (C ₂₂ H ₃₅ N ₅ O ₄ S · 1.1 HCl · 1.0 H ₂ O
As): Calculated: C, 50.45; H, 7.33; N, 13.3
7; Cl, 7.45; Found: C, 50.22; H, 6.97; N, 13.2
9; Cl, 7.36.

Example 4 EtSO ₂ -D-Cha-Pro-4- (NHCH ₂ CH ₂ CH
₂ ) Production of imidazole and HCl

Embedded image _{EtSO 2 -D-Cha-Pro-} 4- (NHCH 2 CH 2 CH
_{2) C 3 H 3 N 2} · HCl Example By methods substantially equivalent to those described in 2, EtSO of _{80mg (80%) 2 -D-} Cha-Pro-4
-(NHCH ₂ CH ₂ CH ₂ ) imidazole · HCl to Et
_{SO 2 -D-Cha-Pro-} 4- (NHCH 2 - trans -
(CHCH) imidazole.HCl. ¹ H-NMR FAB-MS, m / e 468.4 (MH ⁺ ) Elemental analysis (C ₂₂ H ₃₇ N ₅ O ₄ S · 1.1 HCl · 1.0 H ₂ O
As): Calculated: C, 50.26; H, 7.69; N, 13.3
2; Found: C, 50.42; H, 7.31; N, 13.1
5;

Example 5 Preparation of EtSO ₂ -D-Cha-Ohi-4- (NHCH ₂ -trans-CHCH) -imidazole.HCl

Embedded image EtSO ₂ -D-Cha-Ohi-4- (NHCH ₂ -trans-CHCH) -C ₃ H ₃ N ₂ .HCl A) [2S- (2α, 3aβ, 7aβ)]-octahydroindole-2-carboxylic acid Ethyl ester / HCl
Preparation of (Ohi-OEt.HCl) HCl gas was bubbled through a stirred suspension of (S) -indoline-2-carboxylic acid (20 g, 110 mmol) in ethanol (400 mL). When the acid was completely dissolved, the solution was refluxed. After 16 hours, the solution was cooled and the solvent was removed under reduced pressure. The residue was triturated with diethyl ether, then the off-white solid was collected by filtration, washed with hexane and then dried in a vacuum oven at 30 ° C. overnight (25.5 g, 100
%). This solid, (S) -indoline-2-carboxylic acid ethyl ester hydrochloride, was dissolved in ethanol (455 mL). To this was added 5% Pd / C (25.5 g) and the resulting suspension was hydrogenated on a shaker for 8 hours at 4.1 bar. The solution was filtered to remove the catalyst, and the filtrate was concentrated under reduced pressure. The residue was triturated with diethyl ether and the resulting solid was isolated by filtration and 18.8 g
(73%) of a white powder was obtained. ¹ H-NMR FD-MS, m / e 197 (M ⁺ ) Elemental analysis (as C ₁₁ H ₁₉ NO ₂ .HCl): Calculated: C, 56.53; H, 8.63; N, 5.99 Found: C, 56.24; H, 8.44; N, 6.00;

B) EtSO ₂ -D-Phe-Ohi-OEt By a method substantially similar to that described in Example 3-B, 12.3 g (57%) of EtSO ₂ -D-Phe-Oh
i-OEt is converted to EtSO ₂ -D-Phe-OH and HClOh
Prepared from i-OEt. IR ¹ H NMR FD-MS, m / e 436.1 (M ⁺ ) Elemental analysis (as C ₂₂ H ₃₂ N ₂ O ₅ S): Calculated: C, 60.53; H, 7.39; N, 6.42; found: C, 60.62; H, 7.31; N, 6.22;

[0074] _{C) EtSO 2 -D-Phe-} Ohi-OH p- dioxane in (300mL) EtSO ₂ -D-Phe
-OH-OEt added to LiOH.H ₂ O in water (150 mL)
(2.3 g, 55 mmol) was added. After stirring for 16 hours, the solvent was removed under reduced pressure, then the residue was redissolved in water and washed twice with diethyl ether. The aqueous phase was acidified with 5N HCl, the precipitate was filtered, washed with water and dried in vacuo to give 10.1 g (90%) of a pale yellow solid. IR ¹ H-NMR FD-MS, m / e 409.1 (M ⁺ ) Elemental analysis (as C ₂₀ H ₂₈ N ₂ O ₅ S): Calculated: C, 58.80; H, 6.91; N , 6.86; Found: C, 58.57; H, 7.00; N, 6.63.

D) EtSO ₂ -D-Cha-Ohi-OH 8.9 g (95%) of EtSO ₂ -D-Cha-Ohi by a method substantially equivalent to that described in Example 3-D.
The -OH was prepared from _{EtSO 2 -D-Phe-Ohi-} OH. IR ¹ H-NMR FD-MS, m / e 415.3 (MH ⁺ )

E) EtSO ₂ -D-Cha-Ohi-4- (N
HCH ₂ - trans _{-CHCH) EtSO 2 -D-Cha-} Ohi of By methods substantially equivalent to those described in imidazole · HCl Example 3-E, 160mg (49% )
-4-- EtSO a (NHCH ₂ trans -CHCH) imidazole _{· HCl 2 -D-Cha-Ohi} -OH and 4
- prepared from - (NHCH ₂ trans -CHCH) -1-Ts- imidazole · HCl. The product was prepared by preparative RPHPLC (40/60 to 90/10 (A /
B), 150 min). ¹ H-NMR FAB-MS, m / e 520.4 (MH ⁺ ) Elemental analysis (as C ₂₆ H ₄₁ N ₅ O ₄ S.HCl): Calculated: C, 56.15; H, 7.61; N, 12.5
9; Found: C, 56.06; H, 7.69; N, 12.4
4.

Example 6 EtSO ₂ -D-Cha-Ohi-4- (NHCH ₂ CH ₂ CH
₂ ) Production of imidazole and HCl

Embedded image _{EtSO 2 -D-Cha-Ohi-} 4- (NHCH 2 CH 2 CH
_{2) C 3 H 3 N 2} · HCl Example By methods substantially equivalent to those described in _{2, EtSO 2 -D-Cha-} Ohi-4 of 75 mg (92%)
-(NHCH ₂ CH ₂ CH ₂ ) -imidazole.HCl is converted to E
_{tSO 2 -D-Cha-Ohi-} 4- - was prepared from (NHCH ₂ trans -CHCH) imidazole · HCl. ¹ H-NMR FAB-MS, m / e 522.4 (MH ⁺ ) Elemental analysis (C ₂₆ H ₄₃ N ₅ O ₄ S.2.5HCl.1.0H ₂ O
As): Calculated: C, 49.50; H, 7.59; N, 11.1
0; Found: C, 49.89; H, 7.23; N, 11.0.
7.

Example 7 HO ₂ CCH ₂ -D-Cha-Pro-4- (NHCH ₂ -trans-CHCH) imidazole.HCl (N- (carboxymethyl) -D-cyclohexylalanyl-N-
[(E) -3- (Imidazol-4-yl) prop-2
-Enyl] -L-prolinamide hydrochloride)

Embedded image HO ₂ CCH ₂ -D-Cha-Pro-4- (NHCH ₂ -trans-CHCH) C ₃ H ₃ N ₂ .HCl A) Boc-D-Phe-Pro-OBn at 0 ° C. in dichloromethane (600 mL). Boc-D
-Phe-OH (89.1 g, 336 mmol), Pro-
OBn.HCl (81.2 g, 336 mmol), HOB
T (50 g, 370 mmol) and N, N-diisopropylethylamine (176 mL, 1,008 mmol)
1- (3-dimethylaminopropyl) -3-
Ethylcarbodiimide.HCl (71 g, 370 mmol) was added. After stirring for 18 hours, the mixture was diluted with diethyl ether (1 L), then three times with 1N citric acid, once with water (250 mL), three times with saturated aqueous NaHCO ₃ (250 mL), and then with saturated NaCl (250 mL). Washed once. The organic phase is dried (Na ₂ SO ₄ )
Filter and then concentrate under reduced pressure to give 140 g (92.5%) of a pale yellow foam. FD-MS, m / e 452 (M ⁺ ) ¹ H NMR

B) D-Phe-Pro-OBn.HCl While cooling in a 0 ° C. bath, p-dioxane (400 m
HCl gas was bubbled through a stirred solution of Boc-D-Phe-Pro-OBn (74 g, mmol) in L). 1
After 5 minutes, HCl aeration was stopped and the cooling bath was removed. After a further 3 hours, the solvent was removed under reduced pressure. The residue was washed several times with diethyl ether and then dried under reduced pressure to give 61 g (9
8%) of a yellow foam. ¹ H-NMR FD-MS, m / e 353 (MH ⁺ ) Elemental analysis (as C ₂₁ H ₂₄ N ₂ O ₃ .HCl): Calculated: C, 64.86; H, 6.48; N, 7 .20; Found: C, 65.48; H, 6.75; N, 7.94.

C) N- (t-BuO ₂ CCH ₂ ) -N-Boc
-D-Phe-Pro-OBn.HC in DMF (100 mL)
l (20 g, 51 mmol) was added in one portion to a solution of t-butyl bromoacetate (9.9 g, 56 mmol) and N, N-diisopropylethylamine (17.4 mmol) was added.
L, 101 mmol) was added dropwise over 30 minutes. The mixture was stirred at room temperature for 18 hours. Then di-tert-butyl dicarbonate (16.6 g, 76 mmol) and N,
N-diisopropylethylamine (13.2 mL, 76 mmol) was added in one portion and the reaction was then
Stir for 4 hours. The solvent was removed under reduced pressure, and the residue was partitioned between ethyl acetate (1 L) and 1 M aqueous citric acid (500 mL). The layers were separated and the organic phase was once with 1M aqueous citric acid, twice with saturated aqueous sodium bicarbonate and once with brine (50 times each).
(0 mL). The organic phase is dried (Na ₂ S
O ₄ ), filtered and concentrated in vacuo. The amber oil was purified by silica gel chromatography, eluting with an ethyl acetate / hexane gradient (0-30% ethyl acetate / hexane). The fractions containing the product were combined and concentrated to give 19.0 g (66%) of a colorless oil.
The oil slowly crystallized on standing. ¹ H-NMR FD-MS, m / e 566 (M ⁺ ) Elemental analysis (as C ₃₂ H ₄₂ N ₂ O ₇ ): Calculated: C, 67.82; H, 7.47; N, 4.94 Found: C, 68.06; H, 7.33; N, 5.17.

D) N- (t-BuO ₂ CCH ₂ ) -N-Boc
-D-Cha-Pro-OH By a method substantially equivalent to that described in Examples 3-C and 3-D, 22 g (63%) of N- (t-BuO
₂ CCH ₂ ) -N-Boc-D-Cha-Pro-OH
(T-BuO ₂ CCH ₂ ) -N-Boc-Phe-Pro-OBn
Prepared from ¹ H-NMR FD-MS, m / e 483 (M ⁺ ) Elemental analysis (as C ₂₅ H ₄₂ N ₂ O ₇ ): Calculated: C, 62.22; H, 8.77; N, 5.80 Found: C, 62.99; H, 8.96; N, 5.48.

E) HO ₂ CCH ₂ -D-Cha-Pro-4-
(NHCH ₂ -trans-CHCH) imidazole · H
By a method substantially equivalent to that described in Examples 1-H and 1-I, 0.31 g (36%) of HO ₂ CCH
_{2 -D-Cha-Pro-4-} (NHCH 2 - trans -CH
CH) imidazole HCl is converted to N- (t-BuO ₂ CC)
_{H 2) -N-Boc-D} -Cha-Pro-OH and 4-
It was prepared from - (NHCH ₂ trans -CHCH) -1-Ts- imidazole · HCl. The product was prepared by preparative RPHPLC (60 / 40-98 / 2 (A / B),
(150 min). ¹ H-NMR FAB-MS, m / e 432.3 (MH ⁺ ) Elemental analysis (as C ₂₂ H ₃₃ N ₅ O ₄ .1.5HCl): Calculated: C, 54.35; H, 7.15 N, 14.4
0; Found: C, 53.98; H, 7.21; N, 14.0.
3.

Example 8 HO ₂ CCH ₂ -D-Cha-Pro-4- (NHCH ₂ CH ₂
Production of CH ₂ ) imidazole.HCl (N- (carboxymethyl) -D-cyclohexylalanyl-N- [3- (imidazol-4-yl) propyl] -L-prolinamide hydrochloride)

Embedded image _{HO 2 CCH 2 -D-Cha-} Pro-4- (NHCH 2 CH 2
By CH ₂₎ C ₃ H ₃ those described in N ₂ · HCl Example 2 and substantially equivalent methods, HO ₂ CCH ₂ of 0.29g (99%) -D-Cha-
_{Pro-4- (NHCH 2 CH 2} CH 2) C 3 H 3 N 2 · HCl
To HO ₂ CCH ₂ -D-Cha-Pro-4- (NHCH _2-
Prepared from (trans-CHCH) imidazole HCl. ¹ H-NMR FAB-MS, m / e 434.4 (MH ⁺ ) Elemental analysis (as C ₂₂ H ₃₅ N ₅ O ₄ .2.5HCl.1.0 H ₂ O): Calculated: C, 48.69 H, 7.34; N, 12.9
0; Found: C, 48.83; H, 6.99; N, 12.8.
6.

Example 9 D-Cha-Ohi-4- (NHCH ₂ -trans-CHC
H) Production of imidazole / 2HCl

Embedded image D-Cha-Ohi-4- ( NHCH 2 - trans -CHC
_{H) C 3 H 3 N 2} · 2HCl A) 4- (NH 2 CH 2 - trans -CHCH) imidazole · 2HCl TNF (200mL) solution of 4- (Boc ₂ NCH ₂ - trans -CHCH) -1-Ts- Imidazole (4.9 g, 1
(0 mmol) was added HOBT (2.8 g, 20 mmol). After 16 hours, a portion of the solvent was concentrated in vacuo and 1N HCl was added. After stirring for a further 24 hours, the solvent was removed in vacuo, and the residue was
Redissolved in Cl. The aqueous phase was washed several times with n-butanol and once with ethyl acetate. The aqueous phase was then concentrated to give 2 g (100%) of a tan solid. IR ¹ H-NMR FD-MS, m / e 123.0 (M ⁺ )

B) D-Cha-Ohi-4- (NHCH _2-
Trans-CHCH) imidazole.2HCl Examples 5-B, 5-C, 1-H and 1-I (HOBT
By methods substantially equivalent to those described in process is excluded) in, D-Cha-Ohi-4- of 112 mg (NHCH ₂
-Trans-CHCH) imidazole.2HCl to Boc
-D-Cha-OH, HClOhi-OEt and 4- (N
H ₂ CH ₂ - trans -CHCH) imidazole · 2HC
prepared from l. The product is prepared using preparative RPHP
Purified by LC (60 / 40-98 / 2 (A / B), 150 min). IR ¹ H-NMR FD-MS, m / e 428 (M ⁺ ) Elemental analysis (as C ₂₄ H ₃₇ N ₅ O ₂ .3HCl.0.5H ₂ O) Calculated: C, 52.80; H, 7 .57; N, 12.82 Found: C, 53.30; H, 6.90; N, 12.52.

Example 10 D-Cha-Ohi-4- (NHCH ₂ CH ₂ CH ₂ ) imidazole.2HCl (1- (D-cyclohexylalanyl) -N- [3- (imidazol-4-yl) propyl] Production of-[2S- (2α, 3aβ, 7aβ)]-octahydroindole-2-carboxamide dihydrochloride)

Embedded image D-Cha-Ohi-4- ( NHCH 2 CH 2 CH 2) C 3 H 3
_{N 2 · 2HCl A) 4-} HOCH 2 -1-Ts- 4-HOCH 2 in imidazole water (100 mL) - imidazole · H
Cl (18 g, 134 mmol) and K ₂ CO ₃ (55.
(4 g, 400 mmol) in THF (300 mL).
P-Toluenesulfonyl chloride (25.4 g, 1
(34 mmol) was added with vigorous stirring.
After stirring overnight, the solution was partially concentrated under reduced pressure and then extracted three times with ethyl acetate. Then, the ethyl acetate phases were combined, washed twice with brine, dried over MgSO _4, filtered to give a white solid 32 g (95%) then concentrated under reduced pressure. ¹ H-NMR

B) 4-OHC-1-Ts-imidazole At -78 ° C., a stirred solution of oxalyl chloride (8.3 mL, 95 mmol) in dichloromethane (250 mL) was added with DM.
SO (22 mL, 280 mmol) was added dropwise. After 5 min, 4-HOCH ₂ -1 in dichloromethane (50 mL)
A solution of -Ts-imidazole (20 g, 79 mmol) was added via dropping funnel over 2 minutes. 20 minutes later,
Triethylamine (66 mL, 470 mmol) was added, then the cooling bath was removed. After warming the solution to room temperature, water (300 mL) was added and the layers were separated. The aqueous phase was extracted three times with dichloromethane, the combined organic phases were washed twice with brine, then dried over MgSO ₄ , filtered and evaporated under reduced pressure to give 19.8 g (10
(0%).

C) 4-NCCHCH-1-Ts-imidazole To NaH (3.5 g, 87 mmol, 60% dispersed oil) in THF (200 mL) was added 4-OHC-1-Ts in THF (100 mL). -Imidazole (19.8 g, 79
Mmol) and diethylcyanomethylphosphonate (14 g, 79 mmol) were added through a dropping funnel. After 2 hours, the reaction was stopped by adding 1N citric acid. The solvent was evaporated under reduced pressure, then the residue was partitioned between ethyl acetate and 1N citric acid. The organic phase was washed twice with 1N citric acid, once with water and saturated NaHC
Washed twice with O ₃ and once with brine. Then dried the organic phase over MgSO _4, filtered and reduced in volume then concentrated under reduced pressure. Diethyl ether was added and the resulting precipitate was filtered and dried under reduced pressure to 13.1 g (6.
1%) of a white solid was obtained. ¹ H-NMR IR FD-MS, m / e 273 (M ⁺ )

[0089] _{D) 4-NH 2 CH 2} CH 2 CH 2 - 4-NCCHCH-1-Ts- imidazole (2.5g in imidazole · 2HCl THF (50mL), a solution of 9.1 mmol), HO
BT (3.6 g, 2.7 mmol) was added. After stirring overnight, another portion of HOBT (1.2 g, 9.1 mmol) was added and the solution was stirred for 2 hours. The solvent was evaporated under reduced pressure and the residue was partitioned between 1N HCl and ethyl acetate. The layers were then separated, the aqueous phase was washed with butanol, twice with ethyl acetate, and concentrated in vacuo. The residue was dissolved in ethanol (50 mL) and then PtO ₂ (0.11 g) was added. The solution was then placed under a hydrogen atmosphere (5.2 bar) for 4 hours, then filtered through diatomaceous earth, followed by acrodisc (ac
rodisk), and then concentrated under reduced pressure. 1.1 g (75%)
A white solid was obtained. ¹ H-NMR

[0090] E) Boc-D-Cha- Ohi-OH Example 3-B and 5-C to that described in the in substantially the same manner, Boc-D in place of EtSO ₂ -D-Phe-OH Using Cha-OH, Boc-D-Cha-Ohi-
OH was prepared. IR ¹ H-NMR FD-MS, m / e 423 (M ⁺ ) Elemental analysis (as C ₂₃ H ₃₈ N ₂ O ₅ ) Calculated: C, 65.38; H, 9.06; N, 6.63 Found: C, 65.62; H, 9.01; N, 6.55.

F) D-Cha-Ohi-4- (NHCH ₂ C)
_{H 2 CH 2) - Boc-} D-Cha-Ohi-OH in imidazole · 2HCl DMF (15mL)
(1.27 g, 3 _{mmol), 4-NH 2 CH 2} CH 2 CH
₂ -imidazole.2HCl (0.53 g, 3 mmol)
And N, N-diisopropylethylamine (2 mL,
3.3 mmol) of BOP-Cl (1.71).
g, 3.3 mmol). After stirring overnight, the solvent was removed in vacuo was partitioned between ethyl acetate and saturated NH ₄ Cl residue. The organic phase is again saturated with NH
Washed with ₄ Cl, once with water, twice with saturated NaHCO ₃ and once with brine. The solution was dried over MgSO ₄ , filtered under reduced pressure and concentrated. The residue is TFA
(25 mL) and stirred for 2 hours, then the solvent was removed under reduced pressure. The residue was then dissolved in 1N HCl, extracted several times with ethyl acetate, partially concentrated, then prepared by preparative RPHPLC (60 / 40-98 / 2).
(A / B), 150 minutes). Lyophilization of the product-containing fraction resulted in 430 mg (29%) of D-Cha-
_{Ohi-4- (NHCH 2 CH 2} CH 2) - imidazole
2HCl was obtained. IR FD-MS, m / e 430 (MH ⁺ ) Elemental analysis (as C ₂₄ H ₃₉ N ₅ O ₂ .2.8HCl) Calculated: C, 54.22; H, 7.92; N, 13.17 Found: C, 54.63; H, 7.40; N, 12.83.

Example 11 D-Cha-Azt-4- (NHCH ₂ -trans-CHC
H) Imidazole ・ 2HCl

Embedded image D-Cha-Azt-4- ( NHCH 2 - trans -CHC
_{H) C 3 H 3 N 2} · 2HCl

A) Boc-D-Cha-Azt-OH In a manner substantially similar to that described in Examples 1-B, 3-B and 5-C, 4 g of Boc-D-Cha-Azt-OH
-OH was prepared from Azt-OH and Boc-D-Cha-OH. IR ¹ H-NMR FD-MS, m / e 355.2 (MH ⁺ )

B) D-Cha-Azt-4- (NHCH _2-
The trans -CHCH) those described in imidazole · 2HCl Example 9-B in substantially the same way, D-Cha-Azt-4- of 95 mg (NHCH ₂ - trans -CHCH) imidazole · 2HCl Boc-D -Cha
-AZT-OH and 4- (NH ₂ CH ₂ - trans -CH
CH) imidazole.2HCl. The product was purified by preparative RPHPLC (60 / 40-98 /
2 (A / B) for 150 minutes). ¹ H-NMR FD-MS, m / e 360.2 (MH ⁺ ) Elemental analysis (as C ₁₉ H ₂₉ N ₅ O ₂ .3.0HCl 0.5 H ₂ O) Calculated: C, 47.76; 6.96; N, 14.65 Found: C, 74.36; H, 6.69; N, 14.42.

Example 12 D-Cha-Azt-4-NHCH ₂ CH ₂ CH ₂ -imidazole.2HCl

Embedded image D-Cha-Azt-4- NHCH 2 CH 2 CH 2 -C 3 H 3 N 2
· 2HCl D-Cha-Azt- 4-NHCH 2 CH 2 CH 2 - by those described in imidazole · 2HCl Example 10-E in substantially the same way as in the Boc-D-Cha-Ohi- OH Boc- D-Cha-
Azt-OH and 30 mg of D-Cha-Azt-4-N
HCH ₂ CH ₂ CH ₂ - was prepared imidazole · 2HCl. The product was purified by preparative RPHPLC (60 /
40-98 / 2 (A / B), 150 minutes). ¹ H-NMR ES-MS, m / e 362.2 (MH ⁺ ) Elemental analysis (as C ₁₉ H ₃₁ N ₅ O ₂ .3.5HCl.3.0 H ₂ O) Calculated: C, 42.02; H, 7.52; N, 12.89 Found: C, 42.24; H, 7.04; N, 12.74.

Example 13 D-Cha-hPro-4- (NHCH ₂ -trans-CHC
H) Imidazole ・ 2HCl

Embedded image D-Cha-hPro-4- ( NHCH 2 - trans -CHC
H) C ₃ H ₃ N ₂ .2HCl In a manner substantially similar to that described in Example 9,
0.18g of D-Cha-hPro-4- - a (NHCH ₂ trans -CHCH) imidazole · 2HCl hPro-OH
Prepared from The product is prepared by preparative RHPPL
Purified by C (60 / 40-98 / 2 (A / B), 150 minutes). ¹ H-NMR FD-MS, m / e 388.2 (MH ⁺ ) Elemental analysis (as C ₂₁ H ₃₃ N ₅ O ₂ .2HCl.2.5H ₂ O) Calculated: C, 49.90; H, 7.98; N, 13.85 Found: C, 49.67; H, 7.62; N, 13.77.

Example 14 D-Cha-hPro-4-NHCH ₂ CH ₂ CH ₂ -imidazole.2HCl

Embedded image D-Cha-hPro-4- NHCH 2 CH 2 CH 2 -C 3 H 3 N
In substantially the same manner as described in ₂ · 2HCl Example 10, 118 mg of D-Cha-hPro-4- NHCH 2 CH 2
The _{CH 2 -C 3 H 3 N 2} · 2HCl was prepared from hPro-OH. The product was purified by preparative RPHPLC (60 /
40-98 / 2 (A / B), 150 minutes). ¹ H-NMR FD-MS, m / e 390.5 (MH ⁺ ) Analysis of C ₂₁ H ₃₅ N ₅ O ₂ .2.5HCl Calculated: C, 52.47; H, 7.86; N, 14 .57 found: C, 51.92; H, 7.71; N, 14.34 found: C, 53.42; H, 7.68; N, 14.79.

Example 15 HO ₂ CCH ₂ -D-Cha-Pro-4-NHCH ₂ CH _2-
Imidazole 2HCl (N- (carboxymethyl)-
Production of D-cyclohexylalanyl-N- [2- (imidazol-4-yl) ethyl] -L-prolinamide dihydrochloride)

Embedded image _{HO 2 CCH 2 -D-Cha-} Pro-4-NHCH 2 CH 2 -
In substantially the same manner as described in imidazole · 2HCl Example 1-H and 1-I (N-instead of Boc-D-Cha-Pro- OH (t-BuO 2 CCH 2) -N- Boc-D-Cha-
Using DMF instead of Pro-OH and THF,
OBT treatment was omitted), 0.8 g of HO ₂ CCH ₂ -D-
_{Cha-Pro-4-NHCH 2} CH 2 - imidazole · HC
l was prepared from histamine hydrochloride. Final product RP
HPLC (70 / 30-98 / 2 (A / B), 2 hours)
And purified. ^{IR 1 H NMR FAB-MS,} m / e 420.2 (MH +) Elemental analysis _{(C 21 H 33 N 5 O} 4 · 2.1HCl · 0.8H 2 O ) Calculated values: C, 49.41; H, 7.25; N, 13.7
2; Cl, 14.58; Found: C, 49.49; H, 6.89; N, 13.6.
4: Cl, 14.72.

Example 16 Production of EtSO ₂ -D-Cha-Pro-4- (NHCH ₂ CH ₂ ) imidazole.HCl

Embedded image _{EtSO 2 -D-Cha-Pro-} 4- (NHCH 2 CH 2) C
_{3 H 3 N 2 · HCl A} ) Boc-Pro-4- (NHCH 2 CH 2) in substantially the same manner as described in imidazole Example 3-B (using DMF instead of THF), Boc-Pro-
4- (NHCH ₂ CH ₂₎ imidazole Boc-Pro-O
H and histamine.2HCl. ¹ H NMR FD-MS, m / e 309 (MH ⁺ )

B) Boc-Pro-4- (NHCH ₂ CH ₂ )
-1-Ts-imidazole Boc-Pro-4- in dichloromethane (100 mL)
(NHCH ₂ CH ₂₎ imidazole (7.7 g, 25 mmol) stirring solution of p- toluenesulfonyl chloride (4.3 mg, 25 mmol), followed by addition of triethylamine (6.3 mL, 50 mmol). After stirring for 16 hours, the solvent was removed in vacuo, then partitioned between a ethyl acetate and saturated aqueous NH ₄ Cl residue. The organic phase is washed again with a saturated aqueous NH ₄ Cl solution,
It was washed twice with an aqueous solution of O ₃ , filtered, and concentrated under reduced pressure. The residue is subjected to silica gel chromatography,
Elution was performed with a step gradient of 0-20% methanol / chloroform. The product containing fractions were combined and then concentrated under reduced pressure to give 7.65 g (66%) of an off-white solid. ¹ H NMR FAB-MS, m / e 460 (M ⁺ )

C) Pro-4- (NHCH ₂ CH ₂ ) -1-
Ts-imidazole / TFA At 0 ° C., Boc-Pro in dichloromethane (20 mL)
_{-4- (NHCH 2 CH 2) -1} -Ts- imidazole (4.0 g, 8.6 mmol) to a stirred solution of TFA (20
mL) was added. After stirring for 1 hour, the solvent was removed under reduced pressure, and the thick oily residue was washed several times with diethyl ether and dried under reduced pressure to obtain a thick oil. ¹ H-NMR

D) EtSO ₂ -D-Cha-Pro-4- (N
HCH ₂ CH ₂₎ imidazole _{· HCl Pro-4- (NHCH 2} CH 2) -1-Ts- imidazole · TFA (1.43g, 3.0 mmol) in dichloromethane (100 mL) and saturated NaHCO ₃ (100 mL)
Was distributed between The organic phase was separated, dried over MgSO _4, and filtered. EtSO ₂ -D-Cha-Pro
-OH (in substantially the same manner as described in Example 3-D, it was prepared from EtSO ₂ -D-Phe-OH)
(0.79 g, 3.0 mmol) was added followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58 g, 3.0 mmol).
After stirring for 16 hours, the solvent removed and the residue was partitioned between ethyl acetate and saturated aqueous NH ₄ Cl. The organic phase was washed with brine, dried over MgSO _4, and filtered under reduced pressure and concentrated. The residue was dissolved in THF, and HOBT was added to the solution. After stirring for 16 hours, the solvent was removed under reduced pressure, and the residue was partitioned between ethyl acetate and water. The aqueous phase was acidified to pH 2 with 1N HCl and washed four times with ethyl acetate. The aqueous phase was partially concentrated, filtered through an acrodisc, then prepared by preparative RPHPLC (40 / 60-90 / 10 (A /
B), 150 minutes). The product-containing fractions were combined, partially concentrated and then lyophilized to 18
EtSO of _{0mg (12%) 2 -D-} Cha-Pro-4- (N
HCH ₂ CH ₂ ) imidazole.HCl was obtained. ¹ H NMR FD-MS, m / e 454.1 (MH ⁺ ) Elemental analysis (C ₂₁ H ₃₅ N ₅ O ₄ S · 1.3HCl · 1.0H ₂ O
Calculated: C, 48.60; H, 7.44; N, 13.4
9; Cl, 8.88; Found: C, 48.50; H, 7.40; N, 13.5.
0; Cl, 9.20.

Example 17 D-Cha-Pro-4-NHCH ₂ CH ₂ CH ₂ -5-CH ₃
-Production of imidazole / 2HCl

Embedded image D-Cha-Pro-4- NHCH 2 CH 2 CH 2 -5-CH 3
-C ₃ H ₃ N ₂ .2HCl In a manner substantially similar to that described in Example 10, 4-HOCH ₂ -imidazole.HCl was converted to 5-Me-
4-HOCH ₂ -imidazole HCl and Boc-
The D-Cha-Ohi-OH was replaced by Boc-D-Cha-Pro- OH, of 118mg D-Cha-Pro-4 -NHCH 2
The _{CH 2 CH 2 -5-Me-} C 3 H 3 N 2 · 2HCl was prepared. The product was purified by preparative RPHPLC (60 /
40-98 / 2 (A / B), 150 minutes). ¹ H NMR FD-MS, m / e 390 (M ⁺ ) Elemental analysis (as C ₂₁ H ₃₅ N ₅ O ₂ · 3.0 HCl · 1.7H ₂ O) Calculated: C, 47.63; H, 7 .88; N, 13.22 Found: C, 47.98; H, 7.62; N, 12.9.
4.

Example 18 Production of D-Cha-Pro-4-NHCH ₂ CH ₂ CH ₂ CH ₂ -imidazole.2HCl

Embedded image D-Cha-Pro-4- NHCH 2 CH 2 CH 2 CH 2 -C 3
_{H 3 N 2 · 2HCl A)} 4-NCCH 2 CHCH-1-Ts- imidazole DMF (50 mL) solution of 4- (BrCH ₂ - trans -C
HCH) -1-Ts-imidazole (4.5 g, 13.2)
KCN (1.0 g, 15.8 mmol) followed by 18-crown-6 (0.7 g, 2.64 mmol) was added to the stirred solution of (mmol). After stirring overnight, the solvent was removed under reduced pressure, then the residue was dissolved in ethyl acetate and washed with brine. The organic phase is then dried (MgSO ₄ )
Filter and then concentrate under reduced pressure. The residue is ethyl acetate /
Purified by silica gel chromatography, eluting with a hexane gradient. The product containing fractions were combined and concentrated in vacuo to give 1.8 g (47%) of a white solid. ¹ H-NMR

B) D-Cha-Pro-4-NHCH ₂ CH ₂
CH ₂ CH ₂ -imidazole.2HCl 4-NCCHCH-1 was obtained in substantially the same manner as described in Examples 10-D, 10-E and 10-F.
-Ts-imidazole is converted to 4-NCCH ₂ CHCH-1-
After substituting with Ts-imidazole, 130 mg of D-Cha-
_{Pro-4-NHCH 2 CH 2} CH 2 CH 2 -C 3 H 3 N 2 · 2
HCl was prepared. The product is prepared by preparative RPH
PLC (60 / 40-98 / 2 (A / B), 150 minutes)
And purified. ¹ H NMR FD-MS, m / e 390 (M ⁺ ) Elemental analysis (as C ₂₁ H ₃₅ N ₅ O ₂ · 2.0 HCl · 0.8H ₂ O) Calculated: C, 52.89; H, 8 .16; N, 14.68 Found: C, 52.73; H, 7.86; N, 14.8.
One.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C07D 403/12 205 C07D 403/12 205 209 209 (72) Michael Robert Willie, United States 46268 Indiana, United States Langwood Drive 7725, Indianapolis, IN

Claims (3)

[Claims] 1. A compound of the formula (I): X—C (O) —Y—C (O) —NH—CH ₂ —G—Im I wherein X—C (O) — is D-prolinyl, D- _{^{homoprolinyl, R m - (CH 2)}} g -NH-CH 2 -C (O)
-, [Where R ^d is carboxy or methylsulfonyl;
R ^e is NHR ^c , NHCOR ^c or NHCOOR ^c (where R ^c is (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₈ ) cycloalkyl or (C ₃ -C ₈ ) having 4 to 10 carbon atoms. Cycloalkyl- (C ₁ -C ₆ ) alkyl); T is (C ₃ -C ₈ ) cycloalkyl, (C ₁ -C ₈ ) alkyl, _a is 0, 1 or 2; and Q is -OH, (C ₁ -C ₄₎
Alkoxy or —NH—A; A is hydrogen, (C ₁ -C ₄ )
_{Alkyl, R "SO 2 -, R} " OC (O) -, R "C (O)
^{-, R n C (O)} - or ^{_{- (CH 2) g -R m}} ; g is 1,
2 or 3; B is hydrogen or (C ₁ -C ₄ ) alkyl;
R 'is hydrogen or (C ₁ -C ₄₎ alkyl; R "is (C ₁ -
C ₄ ) alkyl, having ₁ to 5 fluorine atoms (C _1-
C _{4) fluoroalkyl, - (CH 2) d -R} m , or unsubstituted or substituted aryl (wherein aryl is phenyl,
Naphthyl, a 5- or 6-membered unsubstituted or substituted aromatic heterocycle having 1 or 2 heteroatoms selected from the same or different sulfur, oxygen and nitrogen, or the same or different selected from sulfur, oxygen and nitrogen 9 or 10 members having one or two heteroatoms
Unsubstituted or substituted fused bicyclic aromatic heterocycle); R
^m is -COOR ^b , -SO ₂ (C ₁ -C ₄ ) alkyl, -S
_{O 3 H, -P (O)} (OR b) 2 or tetrazol -5
- yl; R ⁿ is -COOR ^b or tetrazol-5-yl; each R ^b independently hydrogen or (C ₁ -C ₄₎ alkyl; _d is 1, 2 or 3; ^m is 0, 1 or 2; And Z
Is hydrogen, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkoxy, hydroxy, halo or (C ₁ -C ₄ ) alkylsulfonylamino]; -Y—C (O) — is ] [Where R ^g is (C ₁ -C ₆ ) alkyl, (C ₃ -C ₈ )
Cycloalkyl or _{- (CH 2) p -L- (} CH2) q
-T '; R ^p is (C ₁ -C ₆₎ alkyl, (C ₃ -C ₈₎ cycloalkyl or _{- (CH 2) p -L- (} CH2) q -T'
(Where _p is 0, 1, 2, 3 or 4; L is a single bond,
—O—, —S— or —NH—; _q is 0, 1, 2, or 3; and T ′ is (C ₁ -C ₄ ) alkyl, (C ₃ -C ₈ ) cycloalkyl, —COOH, —CONH ₂ or Ar
(Where Ar is unsubstituted or substituted aryl (where
Aryl is phenyl, naphthyl, the same or different, 5- or 6-membered unsubstituted or substituted aromatic heterocycle having 1 or 2 heteroatoms selected from sulfur, oxygen and nitrogen, or the same or different sulfur, oxygen Or 10-membered unsubstituted or substituted fused bicyclic aromatic heterocycle having one or two heteroatoms selected from nitrogen and nitrogen)); R ^y is —CH ₂ —, —O—, —S—
Or —NH—; and R ^z are a single bond, or R ^y and 3
When two adjacent carbon atoms together form a 5-8 atom saturated carbocyclic ring, one of those atoms is -O
-, - it may be S- or -NH-; and _r is 0, 1 or _{2] G - (CH 2)} s - ( wherein, _s is 0, 1, 2, 3 or 4), or G is _{- (CH 2) t -CH =} CH- ( where, _t is 0, 1 or 2, double bonds bond to Im a trans); and Im have the R at the 5-position Imidazol-4-yl (where R is hydrogen, (C ₁ -C ₄ ) alkyl optionally substituted with a hydroxy substituent or C ₂ -C ₄ (C ₁ -C ₃ ) alkoxy-
(C ₁ -C ₃ ) alkyl);
Each aromatic or heteroaromatic group as described in the definition of independent, or is unsubstituted, halo, hydroxy, (C ₁ -C ₄₎ alkyl, (C ₁ -C ₄₎ alkoxy,
Amino, mono (C ₁ -C ₄ alkyl) amino, di (C _1-
C ₄ alkyl) _{amino, - (CH 2) j COOH} , _{mercapto, -S (O) h (C} 1 -C 4 alkyl), - NHS
(O) _h (C ₁ -C ₄ alkyl), -NHC (O) (C _1-
C _{4 alkyl), - S (O) h} NH 2, -S (O) h NH
(C ₁ -C ₄ alkyl) or —S (O) _h N (C ₁ -C ₄
Alkyl) ₂ (where _h is 0, 1 or 2 and _j is 0, 1, 2, 3 or 4) and independently forms a stable structure with one or two substituents selected from Or a pharmaceutically acceptable salt thereof. 2. A method comprising: a. D-cyclohexylalanyl-N-
[(E) -3- (Imidazol-4-yl) prop-2
-Enyl] -L-prolinamide; b. D-cyclohexylalanyl-N- [3- (imidazol-4-yl) propyl] -L-prolinamide; c. N- (carboxymethyl) -D-cyclohexylalanyl-N-[(E) -3- (imidazol-4-yl) prop-2-enyl] -L-prolinamide; d. N- (carboxymethyl) -D-cyclohexylalanyl-N- [3- (imidazol-4-yl) propyl] -L-prolinamide; e. 1- (D-cyclohexylalanyl) -N- [3-
(Imidazol-4-yl) propyl]-[2S- (2
α, 3aβ, 7aβ) -Octahydroinfall-2
-Carboxamide; and f. The compound according to claim 1, which is selected from N- (carboxymethyl) -D-cyclohexylalanyl-N- [2- (imidazol-4-yl) ethyl] -L-prolinamide; or a pharmaceutically acceptable compound thereof. Salt. 3. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient.